Invasion associated genes from Neisseria meningitidis serogroup B

ABSTRACT

Genes isolated from  Neisseria memingitidis , as well as isolated nucleic acids, probes, expression cassettes, polypeptides, antibodies, immunogenic compositions, antisense nucleic acids, amplification mixtures, and new invasion deficient swains of  Neisseria meningitidis  are provided Methods of detecting  Neisseria meningitidis  and  Neisseria meningitidis  nucleic acids, and methods of inhibiting the invasion of mammalian cells by  Neisseria meningitidis  are also provided.

This is a 35 U.S.C. § 371 national phase application of internationalapplication PCT/U.S.97/19424, filed Oct. 24, 1997, which claimspriority, under 35 U.S.C. § 119(e), of provisional application U.S.application Ser. No. 60/030,432, filed Oct. 24, 1996, the entirecontents; of which are incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to new genes isolated from Neisseria meningitidis.Isolated nucleic acids, probes, expression cassettes, polypeptides,antibodies, immunogenic compositions, antisense nucleic acids,amplification mixtures and new invasion deficient strains of Neisseriameningitidis. The invention also relates to methods of detectingNeisseria meningitidis and Neisseria meningitidis nucleic acids, and tomethods of inhibiting the invasion of mammalian cells by Neisseriameningitidis.

BACKGROUND OF THE INVENTION

Neisseria meningitidis, a Gram-negative encapsulated diplococcus, is anobligate human pathogen and the causative agent of meningococcalmeningitis, one of the most devastating forms of meningitis. Thesebacteria are isolated from humans worldwide and can cause sporadic andepidemic disease. Person-to-person transfer of N. meningitidis occursmainly via the airborne route, and is particularly a problem in placeswhere people are in close quarters, such as prisons, military camps,school class rooms, and day care centers. At any one time, between 2 and10% of individuals in the population carry this organismasymptomatically (Greenfield, S., et al. (1971), J. Infec. Dis.,123:67-73; Moore, P. S., et al. (November 1994), Scientific American,p38-45; Romero, J. D. et al. (1994), Clinical Microbiology Review,7:559-575). With such a high carrier rate, the threat or potential foroutbreaks or epidemics is always present. Although significant advanceshave been made in the area of the pathogenesis of the organism, there ismuch to be learned about the genetics and cell biology of thehost-parasite interaction.

Understanding the mechanism(s) of attachment and invasion is one of themost important aspects in N. meningitidis disease. In order to causedisease, meningacocci must survive and colonize the mucosa of thenasopharynx, pass through these tissue into the bloodstream replicate tolarge numbers in the blood, cross the blood-brain barrier and multiplyin the cerebrospinal fluid (CFS) where they cause inflammation of themeninges. Various models have been used in order to mimic the eventsthat take place during infection in humans. Mouse models (Miller, C. P.(1933), Science, 78:340-341; Holbein, B. E. (1981), Can. J. Microbiol.,27:738-741; Salit, I. E. (1984), Can. J. Microbiol., 30:1022-1029),human nasopharyngeal organ culture (Stephens, D. S., et al. (1991), RevInfect Dis., 13:22-33), chick embryo (Buddingh, G. J. et al. (1987),Science, 86:20.21; Pine, L., et al., Micrbiol. Lett., 130:37-44), andtissue culture monolayer and bilayer systems (Birkness, K. A., et al.(1995), Infect. Immun., 63:402-409) represent some of the modelscommonly used to study virulence of N. meningitidis.

The organ culture system has been used successfully to assess theattachment and invasion properties of various N. meningitidis strains(Salit, I. E. (1984), Can. J. Microbiol., 30:1022-1029).

Designated by serogroup, serological classification of N. meningitidisis based on the capsular polysaccharide composition of the particularstrain. Among the meningococci there are at least thirteen differentserogroups: A, B, C, 29-E, H, I, X, L, W135, X, Y and Z. Of theseserogroups, A, B and C comprise over 90% of the strains isolated frompatients afflicted with meningococcal meningitis (Poolman, J. T., et al.(1995), Infectious Agents and Disease, 4:13-28). The nature of thecapsule in serogroups A and C has led to the development of usefulvaccines against these serogroups. However, the serogroup B capsularpolysaccharide does not induce protection in humans. Many laboratoriesaround the world are concentrating their efforts on the study andcharacterization of epitopes from various membrane and otherextracellular factors for use as vaccine candidates. Some of the mostcommon non-capsule factors in such studies include a number of outermembrane proteins (OMP) such as class 1 (Por A, a cation Specificporin), class 2 or 3 (Pot B, an anion specific protein) and to a lesserextent class 4 and class 5 OMPs (Rmp, and Opc and Opa opacity associatedproteins, respectively). While class 5 Opc and Opa OMPs have been shownto play roles in the invasion of epithelial cells (Virji, M., et al.(1992), Mol. Microbiol., 6;2786-96) due to their antigenic and phasevariability (Aho, E. L.; et al. (1991), Mol. Microbiol., 5:1429-37),they are not considered to be good vaccine candidates.

Class 1 OMPs appear to be good candidates for vaccine studies sincethese proteins have been shown to induce protective immunity. Evaluationof various non-capsular antigens as potential vaccine candidates in invitro bactericidal assays and an infant rat model revealed that class 1OMP had the highest protective capacity compared to factors such as LPSand class 2/3 OMPs (Saukkonen, K., et al. (1989), Vaccine, 7:325-328).However, preliminary data from vaccine trial studies suggests that thesefactors do not elicit a complete immune response, especially in children(Romero. J. D. et al. (1994), Clinical Microbiology Review, 7:559-575;Poolman, J. T., et al. (1995), Infectious Agents and Disease, 4:13-28).The development of fusion or hybrid genes containing epitopes from class1 OMP show great promise as vaccine candidates (Van der Ley, P., et al.(1992), Infect. Immun., 60:3156-3161; Van der Ley, P., et al. (1993),Infect. Immun., 61:4217-4224). However, these hybrids do not elicitprotection in infants, and the immunity induced is type specific andvery short-lived (Poolman, J. T., et al: (1995), Infectious Agents andDisease, 4:13-28). Far these and other reasons, it is or importance toidentify alternative serogroup B vaccine antigens. Initial attachmentand invasion by the pathogen is critical to the disease process. Ifmucosal immunity can be derived against these bacterial factors, thedisease process and the carrier state can be prevented. The presentinvention provides these and other features.

SUMMARY OF THE INVENTION

The invention provides nucleic acids and encoded polypeptides associatedwith invasion of Neisseria meningitidis. The polypeptides are used asdiagnostic reagents as immunogenic reagents; and as components ofvaccines. The nucleic acids are used as diagnostic reagents, ascomponents of vectors and vaccines, and to encode the polypeptides ofthe invention. The invention also provides strains of Neisseriameningitidis which have an invasion deficient phenotype.

In one embodiment, the invention provides isolated nucleic acidsencoding the polypeptides of the invention, including ORF 1 (SEQ IDNO:2), ORF 2 (ORF2 a (SEQ ID NO:4) and ORF2b (SEQ ID NO:5), two separateembodiments depending on alternate start sites for the ORF2polypeptide), ORF 3 (SEQ ID NO:7) and, conservatively modifiedvariations of each of the polypeptides. Exemplar nucleic acids includeSeq 1 (SEQ ID NO:1), Seq 2 (SEQ ID NO:3), and Seq 3 (SEQ ID NO:7) (see,FIGS. 5, 6, and 7 respectively). Other nucleic acids encoding the samepolypeptides include those with silent codon substitutions relative toSeq 1 (SEQ ID NO:1), Seq 2 (SEQ ID NO:3) for Seq 3 (SEQ ID NO:6); aswell as conservatively modified variations thereof.

Isolated nucleic acids which hybridize under stringent conditions to theexemplar nucleic acids Seq 1 (SEQ ID NO:1), Seq 2 (SEQ ID NO:3), or Seq3 (SEQ ID NO:6) are also provided. For example, a complementary nucleicacid to a sequence provided by Seq 1 (SEQ ID NO:1), Seq 2 (SEQ ID NO:3),or Seq 3 (SEQ ID NO:6) hybridizes to Seq 1 (SEQ ID NO:1), Seq 2 (SEQ IDNO:3), or Seq 3 (SEQ ID NO:6), respectively. Nucleic acids which includesubstantial subsequences complementary to Seq 1 (SEQ ID NO:1), Seq 2(SEQ ID NO:3), or Seq 3 (SEQ ID NO:6) also hybridize to Seq 1 (SEQ IDNO:1), Seq 2 (SEQ ID NO:3), or Seq 3 (SEQ ID NO:6), respectively.

Isolated nucleic acids which hybridize under stringent conditions to Seq4 (SEQ ID NO:8) are provided. Seq 4 (SEQ ID NO:8) is a genomic sequencewhich encodes Seq 1 (SEQ ID NO:1), Seq 2 (SEQ ID NO:3), and Seq 3 (SEQID NO:6). Thus, complementary nucleic acids to sequences provided by Seq1 (SEQ ID NO:1), Seq 2 (SEQ ID NO:3), Seq 3 (SEQ ID NO:6), or Seq 4 (SEQID NO:8) all hybridize to Seq 4 (SEQ ID NO:8) under stringentconditions. Similarly, nucleic acids which include substantialsubsequences of Seq 1 (SEQ ID NO:1), Seq 2 (SEQ ID NO:3), Seq 3 (SEQ IDNO:6) or Seq 4 (SEQ ID NO:8) also hybridize to Seq 4 (SEQ ID NO:8). Theisolated nucleic acids are optionally vector nucleic acids whichcomprise a transcription cassette. The transcription cassette optionallyencodes a polypeptide. Typically, the portion of the transcriptioncassette which encodes the polypeptide hybridizes to Seq 4 (SEQ ID NO:8)under stringent conditions. Upon transduction of the transcriptioncassette into a cell, an mRNA which hybridizes to Seq 4 (SEQ ID NO:8)under stringent conditions is produced. The mRNA is translated in thecell into a polypeptide such as the ORF 1 (SEQ ID NO:2), ORF 2a (SEQ IDNO:4), ORF 2b (SEQ ID NO:5) or ORF 3 (SEQ ID NO:7) polypeptides.

Polypeptides encoded by nucleic acids which hybridize under stringentconditions to Seq 4 (SEQ ID NO:8), including Seq 1 (SEQ ID NO:1), Seq 2(SEQ ID NO:3), Seq 3 (SEQ ID NO:7) are provided herein. Exemplarpolypeptides include ORF 1 (SEQ ID NO:1), ORF 2a (SEQ ID NO:4), ORF 2b(SEQ ID NO:5), or ORF 3 (SEQ ID NO:6).

Full length polypeptides of the invention, or antigenic epitopes derivedfrom the full length polypeptides of the invention are optionallypresent in immunogenic compositions. The antigenic epitopes areoptionally incorporated into fusion proteins which optionally includeantigenic epitopes from related or unrelated proteins. The antigenicepitopes are optionally expressed on the surface or antigenic viralvectors.

The immunogenic compositions optionally comprise components to enhanceimmunogenicity, Such as an adjuvant. The compositions optionally includepharmaceutically acceptable excipients. When administered to a mammal,the immunogenic compositions optionally provide an immune responseagainst antigenic epitopes which are included In the immunogeniccompositions. In one preferred embodiment, administration of theimmunogenic composition of the invention to a mammal inhibits invasionof the cells of the mammal by Neisseria meningitidis.

Antibodies which specifically bind to the polypeptides of the inventionare provided. In a preferred embodiment, the antibodies bind to apolypeptide such as ORF 1 (SEQ ID NO:2), ORF 2a (SEQ ID NO:4), ORF 2b(SEQ ID NO:5), or ORF 3 (SEQ ID NO:7); without binding to the E coliFtsZ protein, or to the E coli UNK protein. Typically, the antibodiesspecifically bind to the ORF 1 (SEQ ID NO:2), ORF 2a (SEQ ID NO:4), ORF2b (SEQ ID NO:5), or ORF 3 (SEQ ID NO:7) proteins.

The invention provides isolated Neisseria meningitidis diplococcus. Thediplococcus has a reduced ability to invade tissue culture epithelialcells in vitro as compared to a wild-type Neisseria meningitidisdiplococcus and the genome of the isolated Neisseria meningitidisdiplococcus has a modification in the region of the genome correspondingto Seq 4 (SEQ ID NO:8). In one embodiment, the isolated Neisseriameningitidis diplococcus comprises a transposon insertion in the regionof the genome corresponding to Seq 4 (SEQ ID NO:8).

The invention provides a variety of assays for detecting Neisseriameningitidis, including PCR assays, northern blots, Southern bloc,western blots and ELISA assays. For example, the invention provides PCRreaction mixtures using template nucleic acids which hybridize to Seq 4(SEQ ID NO:8) under stringent conditions. The mixture has a primer pairwhich hybridizes to the template nucleic acid, wherein the primers, whenhybridized to the template, serve as initiation sites for primerextension by a thermostable polymerase such as taq or vent DNApolymerase. The products of PCR amplification are detected by detectingthe amplified nucleic acid products (amplicons) of the PCR reaction.

In several methods relying on nucleic acid hybridization, the detectionof a Neisseria meningitidis nucleic acid in a biological sample isperformed by contacting a probe nucleic acid to the sample and detectingbinding of the nucleic acid to the Neisseria meningitidis nucleic acid.The probe hybridizes to Seq 4 (SEQ ID NO:8), or the complement thereof.Many assay formats are appropriate, including northern and Southernblotting.

In one embodiment, the invention provides methods of inhibiting theinvasion of a mammalian cell by Neisseria meningitidis by expressing ananti-sense RNA molecule in the mammalian cell. The antisense RNAmolecule hybridizes to a nucleic acid which hybridizes under stringentconditions to a nucleic acid encoded by Seq 1 (SEQ ID NO:1), Seq 2 (SEQID NO:3), Seq 3 (SEQ ID NO:7), or Seq 4 (SEQ ID NO:8). Such anti sensemolecules optionally comprise catalytic RNA ribonuclease domains, suchas those derived from a ribozyme.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a schematic of the region from Neisseria meningitidissurrounding the Tn916 transposon from VVV6.

FIG. 2 is a graph of the attachment-invasion assay performed on theHEC-1-B cell line.

FIG. 3 is a graph of the attachment-invasion assay performed on theHEC-1-B cell line with VVV6 and related recombinant Neisseriameningitidis.

FIGS. 4A-4E show the sequence of Seq 4 (SEQ ID NO:8), with ribosomebinding sites (RBS), start sites and stop sites for ORF 1 (SEQ ID NO:7),ORF 2a (SEQ ID NO:4), ORF 2b (SEQ ID NO:5), and ORF 3 (SEQ ID NO:2).

FIGS. 5A-5B show the sequence of Seq 1 (SEQ ID NO:1) (see the nucleicacid sequence of the open reading frame ) and the corresponding aminoacid sequence ORF 1 (SEQ ID NO:2).

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Singleton et al. (1994)Dictionary of Microbiology and Molecular Biology, Second edition, JohnWiley and Sons (New York); Walker (ed) (1988) The Cambridge Dictionaryof Science and Technology, The press syndicate of the University ofCambridge, NY; and Hale and Marham (1991) The Harper Collins Dictionaryof Biology, Harper, Perennial, N.Y. provide one of skill with a generaldictionary of many of the terms used in this invention. Although anymethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present invention, certainpreferred methods and materials are described. For purposes of thepresent invention, the following terms are defined below.

The terms “isolated” or “biologically pure” refer to material which issubstantially or essentially free from components which normallyaccompany it as found in its native state.

The term “nucleic acid” refers to a deoxyribonucleotide orribonucleotide polymer in either single- or double-stranded form, andunless otherwise limited, encompasses known analogues of naturalnucleotides that hybridize to nucleic acids in manner similar tonaturally occurring nucleotides. Unless otherwise indicated, aparticular nucleic acid sequence optionally includes the complementarysequence thereof.

The term “subsequence” in the context of a particular nucleic acidsequence refers to a region of the nucleic acid equal to or smaller thanthe specified nucleic acid. Thus, for example, a viral inhibitor nucleicacid subsequence is a subsequence of a vector nucleic acid, because, inaddition to encoding the viral inhibitor, the vector nucleic acidoptionally encodes other components such as a promoter, a packagingsite, chromosome integration sequences and the like.

Two single-stranded nucleic acids “hybridize” when they form adouble-stranded duplex. The region of double-strandedness can includethe full-length of one or both of the single-stranded nucleic acids, orall of one single stranded nucleic acid and a subsequence of the othersingle stranded nucleic acid or the legion of double-strandedness caninclude a subsequence of each nucleic acid. An overview to thehybridization of nucleic acids is found in Tijssen (1993) LaboratoryTechniques in Biochemistry and Molecular Biology—Hybridization withNucleic Acid Probes part I chapter 2 “Overview of principles ofhybridization and the strategy of nucleic acid probe assays”, Elservier,N.Y.

“Stringent hybridization wash conditions” in the context of nucleic acidhybridization experiments such as Southern and northern hybridizationsare sequence dependent, and are different under different environmentalparameters. An extensive guide to the hybridization of nucleic acids isfound in Tijssen (1993) Laboratory Techniques in Biochemistry andMolecules Biology—Hybridization with Nucleic Acid Probes, part I,chapter 2, “Overview of principles of hybridization and the strategy ofnucleic acid probe assays”, Elsevier, N.Y. Generally, highly stringentwash conditions are selected to be about 5° C. lower than the thermalmelting point (T_(m)) for the specific sequence at a defined ionicstrength and pH. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of the target sequence hybridizes to aperfectly matched probe. Very stringent conditions are selected to beequal to the T_(m) point for a particular probe. Nucleic acids which donot hybridize to each ocher under stringent Conditions are stillSubstantially identical if the polypeptides which they encode aresubstantially identical. This occurs, e.g., when a copy of a nucleicacid is created using the maximum codon degeneracy permitted by thegenetic code.

The term “identical” in the context of two nucleic acid or polypeptidesequences refers to the residues in the two sequences which are the samewhen aligned for maximum correspondence. A nucleic acid is“substantially identical to a reference nucleic acid when it is at leastabout 70% identical, preferably at least about 80% identical, andoptionally about 90% identical or more. When percentage of sequenceidentity is used in reference to proteins or peptides it is recognizedthat residue positions which are not identical often differ byconservative amino acid substitutions, where amino acids residues aresubstituted for other amino acid residues with similar chemicalproperties (e.g., charge or hydrophobicity) and therefore do not changethe functional properties of the molecule. Where sequence differ inconservative substitutions, the percent sequence identity may beadjusted upwards to correct for the conservative nature of thesubstitution. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus for example , where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1 The scoring of conservativesubstitutions is calculated, e.g., according to known algorithm. See,e.g., Meyers and Miller, Computer Applic. Biol. Sci., 4: 11-17 (1988);Smith and Waterman (1981) Adv. Appl. Math. 2: 482; Needleman and Wunsch(1970) J. Mol. Biol. 48: 443; Pearson and Lipman (1988) Proc. Nacl.Aced. Sci. USA 85: 2444; Higgins and Sharp (1988) Gene, 73: 237-244 andHiggins and Sharp (1989) CABIOS 5: 151-153; Corpet, et al. (1988)Nucleic Acids Research 16, 10881-90; Huang, et al. (1992) ComputerApplications in the Biosciences 8, 155-65, and Pearson, et al. (1994)Methods in Molecular Biology 24, 307-31. Alignment is also oftenperformed by inspection and manual alignment.

“Conservatively modified variations” of a particular nucleic acidsequence refers to those nucleic acids which encode identical oressentially identical amino acid sequences, or where the nucleic aciddoes not encode an amino acid sequence, to essentially identicalsequences. Because of the degeneracy of the genetic code, a large numberof functionally identical nucleic acids encode any given polypeptide.For instance, the colons CGU, CGC, CGA, CGG, AGA, and AGG all encode theamino acid arginine. Thus, at every position where an arginine isspecified by a codon, the codon can be altered to any of thecorresponding colons described without altering the encoded polypeptide.Such nucleic acid variations are “silent variations,” which are onespecies of “conservatively modified variations.” Every nucleic acidsequence herein which encodes a polypeptide also describes everypossible silent variation. One of skill will recognize that each colonin a nucleic acid (except AUG, which is ordinarily the only codon formethionine) can be modified to yield a functionally identical moleculeby standard techniques. Accordingly, each “silent variation” of anucleic acid which encodes a polypeptide is implicit in each describedsequence. Furthermore, one of skill will recognize that individualsubstitutions, deletions or additions which alter, add or delete asingle amino acid or a small percentage of amino acids (typically lessthan 5%, more typically less than 1%) in an encoded sequence are“conservatively modified variations” where the alterations result in thesubstitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. The following six groups each containamino acids that are conservative substitutions, for one another.

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenytalanine (F), Tyrosine (Y), Tryptophan (VV).

The term “antibody” refers to a polypeptide substantially encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof. Therecognized immunoglobulin genes include the kappa, lambda, alpha, gamma,delta, epsilon and mu constant region genes, as well as myriadimmunoglobulin variable region genes. Light chains are classified aseither kappa or lambda. Heavy chains are classified as gamma, mu, alpha,delta, or epsilon, which in turn define the immunoglobulin classes, IgG,IgM, IgA, IgD and IgE, respectively.

An exemplar immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies exist e.g., as intact immunoglobulins or as a number of wellcharacterized fragments produced by digestion with various peptidases.Thus, for example; pepsin digests an antibody below the disulfidelinkages in the hinge region to produce F(ab)′₂, a dimer of Fab whichitself is a light chain joined to V_(H)—C_(H)l by a disulfide bond. TheF(ab)′₂ may be reduced under mild conditions to break the disulfidelinkage in the hinge region thereby converting the F(ab)′₂ dimer into anFab′ monomer. The Fab′ monomer is essentially an Fab with part of thehinge region (see, Fundamental Immunology, Third Edition, W. E. Paul,ed., Raven Press, N.Y. (1993), which is incorporated herein byreference, for a more detailed description of other antibody fragments).While various antibody fragments are defined in terms of the digestionof an intact antibody; one of skill will appreciate that such Fab′fragments may be synthesized de novo either chemically or by utilizingrecombinant DNA methodology. Thus, the term antibody, as used herein,also includes antibody fragments either produced by the modification ofwhole antibodies or those synthesized de novo using recombinant DNAmethodologies.

A “chimeric antibody” is an antibody molecule in which (a) the constantregion, or a portion thereof, is altered, replaced or exchanged so thatthe antigen binding site (variable region) is linked to a constantregion of a different or altered class, effector function and/orspecies, or an entirely different molecule which confers new propertiesto the chimeric antibody, e.g., an enzyme, toxin, hormone, growthfactor, drug, etc. or (b) the variable region, or a portion thereof, isaltered, replaced or exchanged with a variable region having a differentor altered antigen specificity.

The term “immunoassay” is an assay that utilizes an antibody tospecifically bind an analyze. The immunoassay is characterized by theuse of specific binding properties of a particular antibody to isolate,target, and/or quantify the analyte.

An “anti-ORF” antibody is an antibody or antibody fragment thatspecifically binds a polypeptide encoded by the Neisseria meningitidisORFs, described herein.

An “expression vector” includes a recombinant expression cassette whichincludes a nucleic acid which encodes a polypeptide which can betranscribed and translated by a cell. A “recombinant expressioncassette” is a nucleic acid construct, generated recombinantly orsynthetically, with a series of specified nucleic acid elements whichpermit transcription of a particular nucleic acid in a target cell. Theexpression vector can be part of a plasmid, virus, or nucleic acidfragment. Typically, the recombinant expression cassette portion of theexpression vector includes a nucleic acid to be transcribed, and apromoter. In some embodiments, the expression cassette also includes,e.g., an origin of replication, and/or chromosome integration elements.A “promoter” is an array of nucleic acid control sequences which directtranscription of a nucleic acid. As used herein, a promoter includesnecessary nucleic acid sequences near the start site of transcription,such as, in the case of a polymerase II type promoter, a TATA element.The promoter also includes distal enhancer or repressor elements whichcan be located as much as several thousand base pairs from the startsite of transcription. A “constitutive” promoter is a promoter which isactive under most environmental conditions and states of development orcell differentiation. An “inducible” promoter responds to anextracellular stimulus. The term “operably linked” refers to functionallinkage between a nucleic acid expression control sequence (such as apromoter or array of transcription factor binding sites) and a secondnucleic acid sequence, wherein the expression control sequence directstranscription of the nucleic acid corresponding to the second sequence.

The term “recombinant” when used with reference to a cell indicates thatthe cell replicates or expresses a nucleic acid, or expresses a peptideor protein encoded by a nucleic acid whose origin is exogenous to thecell. Recombinant cells can express genes that are not found within thenative (non-recombinant) form of the cell. Recombinant cells can alsoexpress genes found in the native form of the cell wherein the genes arere-introduced into the cell by artificial means, for example under thecontrol of a heterologous promoter.

An “immunogenic composition” is a composition which elicits theproduction of an antibody which binds a component of the compositionwhen administered to a mammal, or which elicits the production of acell-mediated immune response against a component of the composition.

An “antigenic epitope” in the context of a polypeptide is a polypeptidesubsequence which, when presented as an immunogen, or as a portion of animmunogen (e.g., with a carrier protein or adjuvant or on the surface ofa vital vector), elicits an antibody which specifically binds to thefull length polypeptide.

DETAILED DESCRIPTION OF THE INVENTION

Using several new tools and techniques, the identification of bacterialgene(S) which are Involved in the process of cell adhesion and invasionare described. A Tn916-mutant library of N. meningitidis, serogroup B,strain NMB (Kathariou, S., et al. Mol. Microbiol., 4:729-735), wasexamined for the lost ability to attach or invade tissue cultureepithelial cells (HEC1-B). Several hundred mutants were screened, andone strain, VVV6, showed a significant >10-fold decrease in its abilityto associate with the HEC1-B monolayer, compared to its parent strain;NMB. Southern hybridization, polymerase chain reaction, and DNA sequenceanalysis data revealed the presence of a single intact, Class 1, copy oftransposon Tn916. To demonstrate linkage between the transposoninsertion site and mutant phenotype backtransformants were created viahomologous recombination. All seven recombinants also showed aninvasion-deficient phenotype as observed with VVV6: Nucleotide sequenceanalysis shows that the Tn916 insertion occurred between two openreading frames (ORFs). The nature or function of the products encoded bythese ORFs is not known: ORF 3 (SEQ ID NO:7) shows no significanthomology any known gene, while ORF 2(ORF 2a (SEQ ID NO:4); ORF 2b (SEQID NO:5)) shows 60% identity to an E. coli gene with no known function.Adjacent to ORF 2, an open reading frame encoding ORF 1 was found. ORF 1is the Neisseria meningitidis frz gene homologue.

Making Neisseria meningitidis Nucleic Acids and Polypeptides

Several specific nucleic acids encoding Neisseria meningitidispolypeptides are described herein. These nucleic acids can be made usingstandard recombinant or synthetic techniques. Given the nucleic acids ofthe present invention, one of skill can construct a variety of clonescontaining functionally equivalent nucleic acids, such as nucleic acidswhich encode the same polypeptide. Cloning methodologies to accomplishthese ends, and sequencing methods to verify the sequence of nucleicacids are well known in the art. Examples of appropriate cloning andsequencing techniques, and instructions sufficient to direct persons ofskill through many cloning exercises are found in Berger and Kimmel,Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et al. (1989)Molecular Cloning—A Laboratory Manual (2nd ed.) Vol. 1-3; and CurrentProtocols in Molecular Biology, F. M. Ausubel et al., eds., CurrentProtocols, a joint venture between Greene Publishing Associates, Inc.and John Wiley & Sons, Inc., (1994 Supplement) (Ausubel). Productinformation from manufacturers of biological reagents and experimentalequipment also provide information useful in known biological methods.Such manufacturers include the SIGMA chemical company (Saint Louis,Mo.), R&D systems (Minneapolis, Minn.), Pharmacia LKB Biotechnology(Piscataway, N.J.), CLONTECH Laboratories, Inc. (Palo Alto, Calif.),Chem Genes Corp., Aldrich. Chemical Company (Milwaukee, Wis.), GlenResearch, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersberg, Md.),Fluky Chemica-Biochemika Analytika (Fluky Chemie AG, Buchs,Switzerland), Invitrogen, San Diego, Calif., and Applied Biosystems(Foster City, Calif.), as well as many other commercial sources known toone of skill.

The nucleic acid compositions of this invention, whether RNA, cDNA,genomic DNA, or a hybrid of the various combinations, are isolated frombiological sources or synthesized in vitro. The nucleic acids of theinvention are present in transformed or transfected cells, intransformed or transfected cell lysates, or in a partially purified orsubstantially pure form.

In vitro amplification techniques suitable for amplifying sequences foruse as molecular probes or generating nucleic acid fragments for,subsequent subcloning are known. Examples of techniques sufficient todirect persons of skill through such in vitro amplification methods,including the polymerase chain reaction (PCR) the ligase chain reaction(LCR), Qβ-replicase amplification and other RNA polymerase mediatedtechniques (e.g., NASBA) are found in Bergen, Sambrook et al. (1989)Molecular Cloning-A Laboratory Manual (2nd Ed) Vol. 1-3; and Ausubel, aswell as Mullis et al., (1987) U.S. Pat. No. 4,683,202; PCR Protocols AGuide to Methods and Applications (Innis et al. eds) Academic Press Inc.San Diego, Calif. (1990) (Innis); Arnheim & Levinson (Oct. 1, 1990) C&EN36-47; The Journal Of NIH Research (1991) 3, 81-94; (Kwoh et al. (1989)Proc. Natl. Acad. Sci. USA 86, 1173; Guatelli et al. (1990) Proc. Natl.Acid. Sci. USA 87, 1874; Lomell et al. (1989) J. Clin. Chem 35, 1826;Landegren et al., (1988) Science 241, 1077-1080; Van Brunt (1990)Biotechnology 8, 291-294; Wu and Wallace, (1989) Gene 4, 560; Barringeret al. (1990) Gene 89, 117, and Sooknanan and Malek (1995) Biotechnology13: 563-564. Improved methods of cloning in vitro amplified nucleicacids are described in Wallace et al., U.S. Pat. No. 5,426,039. Improvedmethods of amplifying large nucleic acids are summarized in Cheng et al.(1994) Nature 369: 684-685 and the references therein. One of skill willappreciate that essentially any RNA can be converted into a doublestranded DNA suitable for restriction digestion, PCR expansion aidsequencing using reverse transcriptase and a polymerase. See, Ausbel,Sambrook and Bergen, all supra.

Oligonucleotides for use as probes, e.g., in in vitro Neisseriameningitidis nucleic acid amplification methods, or for use as nucleicacid probes to detect Neisseria meningitidis nucleic acids are typicallysynthesized chemically according to the solid phase phosphoramiditetriester method described by Beaucage and Caruthers (1981), TehrahedronLetts., 22(20):1859-1862, e.g., using an automated synthesizer, e.g., asdescribed in Needham-VanDevanter et al. (1984) Nucleic Acids Res.,12:6159-6168. Oligonucleotides can also be custom made and ordered froma variety of commercial sources known to persons of skill. Purificationof oligonucleotides, where necessary, is typically performed by eithernative acrylamide gel electrophoresis or by anion-exchange HPLC asdescribed in Pearson and Regnier (1983) J. Chrom. 255:137-149. Thesequence of the synthetic oligonucleotides can be verified using thechemical degradation method of Maxam and Gilbert (1980) in Grossman andMoldave (eds.) Academic Press, New York, Methods in Enzymology65:499-560.

One of skill will recognize many ways of generating alterations in agiven nucleic acid sequence. Such well-known methods includesite-directed mutagenesis, PCR amplification using degenerateoligonucleotides, exposure of cells containing the nucleic acid tomutagenic agents or radiation, chemical synthesis of a desiredoligonucleotide (e.g., in conjunction with ligation and/or cloning togenerate large nucleic acids) and other well-known techniques. See,Giliman and Smith (1979) Gene 8:81-97, Roberts et al. (1987) Nature328:731-734 and Sambrook, Innis, Ausbel, Bergen, Needham VanDevanter andMullis (all supra).

Polypeptides of the invention are optionally synthetically prepared in awide variety of well-known ways. Polypeptides of relatively short sizeare typically synthesized in solution or on a solid support inaccordance with conventional techniques. See, e.g., Merrifield (1963) J.Am. Chem. Soc. 85:2149-2154. Various automatic synthesizers andsequencers are commercially available and can be used in accordance withknown protocols. See, e.g., Stewart and Young (1984) Solid Phase PeptideSynthesis, 2d. ed., Pierce Chemical Co. Polypeptides are also producedby recombinant expression of a nucleic acid encoding the polypeptidefollowed by purification using standard techniques. Solid phasesynthesis in which the C-terminal amino acid of the sequence is attachedto an insoluble support followed by sequential addition of the remainingamino acids in the sequence is the preferred method for the chemicalsynthesis of the polypeptides of this invention. Techniques for solidphase synthesis are described by Barany and Merrifield, Solid-PhasePeptide Synthesis; pp. 3-284 in The Peptides: Analysis, Synthesis,Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A.,Merrifield, et al. J. Am. Chem. Soc., 85: 2149-2156 (1963), and Stewartet al., Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co.,Rockford, Ill. (1984).

Cloning and Expressing Neisseria meningitidis Nucleic Acids

In a preferred embodiment, the polypeptides, or subsequences thereof,are synthesized using recombinant DNA methodology. Generally, thisinvolves creating a DNA sequence that encodes the protein, placing theDNA in an expression cassette under the control of a particularpromoter, expressing the protein in a host cell, isolating the expressedprotein and, if required, renaturing the protein.

Once a nucleic acid encoding a polypeptide of the invention is isolatedand cloned, the nucleic acid is optionally expressed in a recombinantlyengineered cells known to those of skill in the art. Examples of suchinclude bacteria, yeast, plant, filamentous fungi, insect (especiallyemploying baculoviral vectors) and mammalian cells. The recombinantnucleic acids are operably linked to appropriate control sequences forexpression in the selected host. For E. coli, example control sequencesinclude the T7, trp, or lambda promoters, a ribosome binding site andpreferably a transcription termination signal. For eukaryotic cells, thecontrol sequences typically include a promoter and preferably anenhancer derived from immunoglobulin genes, SV40, cytomegalovirus, etc.,and a polyadenylation sequence, and may include splice donor andacceptor sequences.

The plasmids of the invention Can be transferred into the chosen hostcell by well-known methods such as Calcium Chloride transformation forE. coli and calcium phosphate treatment or electroporation for mammaliancells. Cells transformed by the plasmids can be selected by resistanceto antibiotics conferred by genes contained on the plasmids, such as theamp, gpt, neo and hyg genes.

Once expressed, the recombinant polypeptides can be purified accordingto standard procedures of the art, including ammonium sulfateprecipitation, affinity columns, column chromatography, gelelectrophoresis and the like (see, generally, R. Scopes, PolypeptidePurification, Springer-Verlag, N.Y. (1982), Deutscher, Methods inEnzymology Vol. 182: Guide to Polypeptide Purification, Academic Press,Inc. N.Y. (1990)). Once purified, partially or to homogeneity asdesired, the polypeptides may then be used (e.g., as immunogens forantibody production).

After chemical synthesis, biological expression, or purification, thepolypeptides) may possess a conformation substantially different thanthe native conformations of the constituent polypeptides. In this case,it is helpful to denature and reduce the polypeptide and then to causethe polypeptide to re-fold into the preferred conformation. Methods ofreducing and denaturing polypeptides and inducing re-folding are wellknown to those of skill in the art (See, Debinski et al. (1993). J.Biol. Chem., 268: 14065-14070; Kreitman and Pastan (1993) Bioconjug.Chem., 4: 581-585; and Buchner, et al., (1992) Anal. Biochem.,205:263-270). Debinski et al., for example, describe the denaturationand reduction of inclusion body polypeptides in guanidine-DTE Thepolypeptide is then refolded in a redox buffer containing oxidizedglutathione and L-arginine.

One of skill will recognize that modifications can be made to thepolypeptides without diminishing their biological activity. Somemodifications may be made to facilitate the cloning, expression orincorporation of the targeting molecule into a fusion polypeptide. Suchmodifications are well known to those of skill in the art and include,for example, a methionine added at the amino terminus to provide aninitiation site, or additional amino acids (e.g., poly His) placed oneither terminus to create conveniently located restriction sites ortermination colons or purification sequences.

Making Conservative Modifications of the Nucleic Acids and Polypeptidesof the Invention

One of skill will appreciate that many conservative variations of thenucleic acid and polypeptide sequences of the figures and sequencelistings yield functionally identical products. For example, due to thedegeneracy of the genetic code, “silent substitutions” (i.e.,substitutions of a nucleic acid sequence which do not result in analteration in an encoded polypeptide) are an implied feature of everynucleic acid sequence which encodes an amino acid. Similarly,“conservative amino acid substitutions.” in one or a few amino acids inan amino acid sequence are substituted with different amino acids withhighly similar properties (see, the definitions section, supra), arealso readily identified as being highly similar to a disclosed aminoacid sequence, or to a disclosed nucleic acid sequence which encodes anamino acid. Such conservatively Substituted variations of eachexplicitly listed sequence are a feature of the present invention.

One of skill will recognize many ways of generating alterations in agiven nucleic acid sequence. Such well-known methods includesite-directed mutagenesis, PCR amplification using degenerateoligonucleotides, exposure of cells containing the nucleic acid tomutagenic agents or radiation, chemical synthesis of a desiredoligonucleotide (e.g., in conjunction with ligation and/or cloning togenerate large nucleic acids) and other well-known techniques. See,Giliman and Smith (1979) Gene 8:81-97, Roberts et al. (1987) Nature328:731-734 and Sambrook, Innis, Ausbel, Bergen, Needham VanDevanter andMullis (all supra).

Most commonly, polypeptide sequences are altered by changing thecorresponding nucleic acid sequence and expressing the polypeptide.However, polypeptide sequences are also optionally generatedsynthetically using commercially available peptide synthesizers toproduce any desired polypeptide (see, Merrifield, and Stewart and Young,supra).

One of skill can select a desired nucleic acid or polypeptide of theinvention based upon the sequences provided and upon knowledge in theart regarding proteins generally. Knowledge regarding the nature ofproteins and nucleic acids allows one of skill to select appropriatesequences with activity similar or equivalent to the nucleic acids andpolypeptides disclosed in the sequence listings herein. The definitionssection herein describes exemplar conservative amino acid substitutions.

Finally, most modifications to nucleic acids and polypeptides areevaluated by routine screening techniques in suitable assays for thedesired characteristic. For instance, changes in the immunologicalcharacter of a polypeptide can be detected by an appropriateimmunological assay. Modifications of other properties such as nucleicacid hybridization to a target nucleic acid, redox or thermal stabilityof a protein, hydrophobicity, susceptibility to proteolysis, or thetendency to aggregate are all assayed according to standard techniques.

Screening for Neisseria meningitidis Nucleic Acids and the Use ofNeisseria meningitidis Nucleic Acids as Molecular Probes

The nucleic acids of the invention are useful as molecular probes, inaddition to their utility in encoding the polypeptides described herein.A wide variety of formats and labels are available and appropriate fornucleic acid hybridization, including those reviewed in Tijssen (1993)Laboratory Techniques in biochemistry and molecularbiology-hybridization with nucleic acid probes parts I and II, Elsevier,N.Y. and Choo (ed) (1994) Methods In Molecular Biology Volume 33—In SituHybridization Protocols Humana Press Inc., New Jersey (see also, otherbooks in the Methods in Molecular Biology series); see especially,Chapter 21 of Choo (id) “Detection of Virus Nucleic Acids by Radioactiveand Nonisotopic in Situ Hybridization”

For instance, PCR, LCR, and other amplification techniques (see, supra)are routinely used to detect Neisseria meningitidis nucleic acids inbiological samples. Accordingly, in one class of embodiments; thenucleic acids of the invention are used as primers of templates, or aspositive controls in amplification reactions for the detection ofNeisseria meningitidis in a biological samples such as cerebrospinalfluid. Briefly, nucleic acids with sequence identity or complementarityto Seq 4 (SEQ ID NO:8), or the complement thereof are used as templatesto synthetically produce oligonucleotides of about 15-23 nucleotideswith sequences similar or identical to the complement of a selectedNeisseria meningitidis nucleic acid subsequence. The oligonucleotidesare then used as primers in amplification reactions such as PCR todetect selected Neisseria meningitidis nucleic acids in biologicalsamples, such as a cerebrospinal fluid extract. A nucleic acid of theinvention (i.e., a cloned nucleic acid corresponding to the region to beamplified) is also optionally used as an amplification template in aseparate reactions as a positive control to determine that theamplification reagents and hybridization conditions are appropriate.

Other methods for the detection of nucleic acids in biological samplesusing nucleic acids of the invention include Southern blots, northernblots, in situ hybridization (including Fluorescent in situhybridization (FISH), and a variety of other techniques overviewed inChoo (supra)). A variety of automated solid-phase detection techniquesare also appropriate. For instance, very large scale immobilized polymerarrays (VLSIPS™) are used for the detection of nucleic acids. See,Tijssen (supra), Fodor et al. (1991) Science, 251: 767-777; Sheldon etal. (1993) Clinical Chemistry 39(4): 718-719 and Kozal et al. (1996)Nature Medicine 2(7): 753-759.

Antibodies to selected Neisseria meningitidis ORF polypeptide(s).

Antibodies are raised to selected Neisseria meningitidis ORFpolypeptides of the present invention, including individual, allelic,strain, or species variants, and fragments thereof, both in theirnaturally occurring (full-length) forms and in recombinant forms.Additionally, antibodies are raised to these polypeptides in eithertheir native configurations or in non-native configurations.Anti-idiotypic antibodies can also be generated. Many methods of makingantibodies are known to persons of skill. The following discussion ispresented as a general overview of the techniques available; however,one of skill will recognize that many variations upon the followingmethods are known.

A number of immunogens are used to produce antibodies specificallyreactive with Neisseria meningitidis ORF 1 (SEQ ID NO:2), ORF 2a (SEQ IDNO:4), ORF 2b (SEQ ID NO:5), or ORF 3 (SEQ ID NO:7) polypeptides.Recombinant or synthetic polypeptides of 10 amino acids in length, orgreater, typically 20 amino acids in length, or greater, more typically30 amino acids in length, or greater, selected from amino acidsub-sequences of ORF 1 (SEQ ID NO:2), ORF 2a (SEQ ID NO:4), ORF 2b (SEQID NO:5), or ORF 3 (SEQ ID NO:7) are the preferred polypeptide immunogenfor the production of monoclonal or polyclonal antibodies. In one classof preferred embodiments, an immunogenic peptide conjugate is alsoincluded as an immunogen. Naturally occurring polypeptides are also usedeither in pure or impure form. An antigenic domain is ordinarily atleast about 3 amino acids in length often at least about 5 amino acidsin length, generally at least about 9 amino acids in length and often atleast about 15 amino acids in length. The antigenic domain ordinarilyincludes the binding site for an antibody, which typically vary from 3to about 20 amino acids in length, and which are generally about 8 to 12amino acids in length.

Recombinant polypeptides are expressed in eukaryotic or prokaryoticcells and purified using standard techniques. The polypeptide, or asynthetic version thereof, is then injected into an animal capable ofproducing antibodies. Either monoclonal or polyclonal antibodies can begenerated for subsequent use in immunoassays to measure the presence andquantity of the polypeptide.

Methods of producing polyclonal antibodies are known to those of skillin the art. In brief, an immunogen (antigen), preferably a purifiedpolypeptide, a polypeptide coupled to an appropriate carrier (e.g., GST,keyhole limpet hemanocyanin, etc.), or a polypeptide incorporated intoan immunization vector such as a recombinant vaccinia virus (see, U.S.Pat. No. 4,722,848) is mixed with an adjuvant and animals are immunizedwith the mixture. The animal's immune response to the immunogenpreparation is monitored by taking test bleeds and determining the titerof reactivity to the polypeptide of interest. When appropriately hightiters of antibody to the immunogen are obtained, blood is collectedfrom the animal and antisera are prepared. Further fractionation of theantisera to enrich for antibodies reactive to the polypeptide isperformed where desired (see, e.g., Coligan (1991) Current Protocols inImmunology Wiley/Greene, NY; and Harlow and Lane (1989) Antibodies: ALaboratory Manual Cold Spring Press, NY).

Antibodies, including binding fragments and single chain recombinantversions thereof, against whole or predetermined fragments of selectedNeisseria meningitidis ORFs are raised by immunizing animals, e.g., withconjugates of the fragments with carrier proteins as described above.Typically, the immunogen of interest is a peptide of at least about 10amino acids, more typically the peptide is 20 amino acids in length,generally the fragment is 25 amino acids in length and often thefragment is 30 amino acids in length or greater. The peptides areoptionally coupled to a carrier protein (e.g., as a fusion protein), orare recombinantly expressed in an immunization vector. Antigenicdeterminants on selected Neisseria meningitidis ORF peptides to whichantibodies bind are typically 3 to 10 amino acids in length.

Monoclonal antibodies are prepared from cells secreting the desiredantibody. These antibodies are screened for binding to normal ormodified polypeptides or screened for agonistic or antagonisticactivity, e.g., activity mediated through a selected Neisseriameningitidis ORF polypeptide. Specific monoclonal and polyclonalantibodies will usually bind with a K_(D) of at least about 0.1 mM, moreusually at least about 50 μM, and preferably at least about 1 μM orbetter.

In some instances, it is desirable to prepare monoclonal antibodies fromvarious mammalian hosts, such as mice, rodents, primates, humans, etc.Description of techniques for preparing such monoclonal antibodies arefound in, e.g., Stites et al. (eds.) Basic and Clinical Immunology (4thed.) Lange Medical Publications, Los Altos, Calif., and references citedtherein; Harlow and Lane, Supra; Goding (1986) Monoclonal Antibodies:Principles and Practice (2d ed.) Academic Press, New York, N.Y.; andKohler and Milstein (1975) Nature 256: 495-497. Summarized briefly, thismethod proceeds by injecting an animal with an immunogen. The animal isthen sacrificed and cells taken from its spleen, which are fused withmyeloma cells. The result is a hybrid cell or “hybridoma” that iscapable of reproducing in vitro. The population of hybridomas is thenscreened to isolate individual clones, each of which secrete a singleantibody species to the immunogen. In this manner, the individualantibody species obtained are the products of immortalized and clonedsingle B cells from the immune animal generated in response to aspecific site recognized on the immunogenic substance.

Alternative methods of immortalization include transformation withEpstein Barr Virus, oncogenes, or retroviruses, or other methods knownin the art. Colonies arising from single immortalized cells are screenedfor production of antibodies of the desired specificity and affinity forthe antigen, and yield of the monoclonal antibodies produced by suchcells is enhanced by various techniques, including injection into theperitoneal cavity of a vertebrate (preferably mammalian) host. Thepolypeptides and antibodies of the present invention are used with orwithout modification, and include chimeric antibodies such as humanizedmurine antibodies.

Other suitable techniques involve selection of libraries of recombinantantibodies in phage or similar vectors (see, e.g., Huse et al. (1989)Science 246: 1275-1281; and Ward, et al. (1989) Nature 341: 544-546; andVaughan et al. (1996) Nature Biotechnology, 14: 309-314).

Frequently, the polypeptides and antibodies will be labeled by joining,either covalently or non-covalently, a substance which provides for adetectable signal. A wide variety of labels and conjugation techniquesare known and are reported extensively in both the scientific and patentliterature. Suitable labels include radionucleotides, enzymes,substrates, cofactors, inhibitors, fluorescent moieties,chemiluminescent moieties, magnetic particles, and the like. Patentsteaching the use of such labels include U.S. Pat. Nos. 3,817,837;3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.Also, recombinant immunoglobulins may be produced. See, Cabilly, U.S.Pat. No. 4,816,567; and Queen et al. (1989) Proc. Nat'l Acad. Sci. USA86: 10029-10033.

The antibodies of this invention are also used for affinitychromatography in isolating natural or recombinant Neisseriameningitidis ORF polypeptides. Columns are prepared, e.g., with theantibodies linked to a solid support, e.g., particles, such as agarose,Sephadex, or the like, where a cell lysate is passed through the column,washed, and treated with increasing concentrations of a mild denaturant,whereby purified polypeptides are released.

The antibodies can be used to screen expression libraries for particularexpression products such as normal or abnormal Neisseria meningitidisORF polypeptides, or for related polypeptides related to a selectedNeisseria meningitidis ORF polypeptide. Optionally, the antibodies insuch a procedure are labeled with a moiety allowing easy detection ofpresence of antigen by antibody binding.

Antibodies raised against polypeptides can also be used to raiseanti-idiotypic antibodies. These are useful for detecting or diagnosingvarious pathological conditions related to the presence of therespective antigens.

The antibodies of this invention can also be administered to an organism(e.g., a human patient) for therapeutic purposes (e.g., to blockinfection by Neisseria meningitidis, or as targeting molecules whenconjugated or fused to effector molecules such as labels, cytotoxins,enzymes, growth factors, drugs, etc.). Antibodies administered to anorganism other than the species in which they are raised can beimmunogenic. Thus, for example, murine antibodies administered to ahuman can induce an immunologic response against the antibody (e.g., thehuman anti-mouse antibody (HAMA) response), particularly after multipleadministrations. The immunogenic properties of the antibody are reducedby altering portions, or all, of the antibody into characteristicallyhuman sequences thereby producing chimeric, or human, antibodiesrespectively.

Humanized (chimeric) antibodies are immunoglobulin molecules comprisinga human and non-human portion. The antigen combining region (or variableregion) of a humanized chimeric antibody is derived from a non-humansource (e.g., murine) and the constant region of the chimeric antibody(which confers biological effector function, such as cytotoxicity, tothe immunoglobulin) is derived from a human source. The humanizedchimeric antibody has the antigen binding specificity of the non-humanantibody molecule and the effector function conferred by the humanantibody molecule. A large number of methods of generating chimericantibodies are well known to those of skill in the art (see, e.g., U.S.Pat. Nos.: 5,502,167, 5,500,362, 5,491,088, 5,482,856, 5,472,693,5,354,847, 5,292,867, 5,231,026, 5,204,244, 5,202,238, 5,169,939,5,081,235, 5,075,431, and 4,975,369).

In general, the procedures used to produce these chimeric antibodiesconsist of the following steps (the order of some stepsinterchangeable): (a) identifying and cloning the correct gene segmentencoding the antigen binding portion of the antibody molecule; this genesegment (known as the VDJ, variable, diversity and joining regions forheavy chains or VJ, variable, joining regions for light chains (orsimply as the V or Variable region) may be in either the cDNA or genomicform; (b) cloning the gene segments encoding the constant region ordesired part thereof; (c) ligating the variable region with the constantregion so that the complete chimeric antibody is encoded in atranscribable and translatable form; (d) ligating this construct into avector containing a selectable marker and gene control regions such aspromoters, enhancers and poly(A) addition signals; (e) amplifying thisconstruct in a host cell (e.g., bacteria); and, (f) introducing the DNAinto eukaryotic cells (transfection) most often mammalian lymphocytes.

Antibodies of several distinct antigen binding specificities have beenmanipulated by these protocols to produce chimeric proteins (e.g.,anti-TNP: Boulianne et al. (1984) Nature, 312: 643; and anti-tumorantigens: Sahagan et al. (1986) J. Immunol., 137: 1066). Likewise,several different effector functions have been achieved by linking newsequences to those encoding the antigen binding region. Some of theseeffectors include enzymes (Neuberger et al. (1984) Nature 312: 604),immunoglobulin constant regions from another species, and constantregions of another immunoglobulin chain (Sharon et al. (1984) Nature309: 364; Tan et al., (1985) J. Immunol. 135: 3565-3567).

In one preferred embodiment, a recombinant DNA vector is used totransfect a cell line that produces an antibody. The novel recombinantDNA vector contains a “replacement gene” to replace all or a portion ofthe gene encoding the immunoglobulin constant region in the cell line(e.g., a replacement gene may encode all or a portion of a constantregion of a human immunoglobulin, a specific immunoglobulin class, or anenzyme, a toxin, a biologically active peptide, a growth factor,inhibitor, or a linker peptide to facilitate conjugate to a drug, toxin,or other molecule, etc.), and a “target sequence” which allows fortargeted homologous recombination with immunoglobulin sequences withinthe antibody producing cell.

In another embodiment, a recombinant DNA vector is used to transfect acell line that produces an antibody having a desired effector function,(e.g., a constant region of a human immunoglobulin) in which case, thereplacement gene contained in the recombinant vector may encode all or aportion of a region of an antibody and the target sequence contained inthe recombinant vector allows for homologous recombination and targetedgene modification within the antibody producing cell. In eitherembodiment, when only a portion of the variable or constant region isreplaced, the resulting chimeric antibody may define the same antigenand/or have the same effector function yet be altered or improved sothat the chimeric antibody may demonstrate a greater antigenspecificity, greater affinity binding constant, increased effectorfunction, or increased secretion and production by the transfectedantibody producing cell line, etc. Regardless of the embodimentpracticed, the processes of selection for integrated DNA (via aselectable marker), screening for chimeric antibody production, and cellcloning, can be used to obtain a clone of cells producing the chimericantibody.

Thus, a piece of DNA which encodes a modification for a monoclonalantibody can be targeted directly to the site of the expressedimmunoglobulin gene within a B-cell or hybridoma cell line. DNAconstructs for any particular modification may be used to alter theprotein product of any monoclonal cell line or hybridoma. Such aprocedure circumvents the task of cloning both heavy and light chainvariable region genes from each B-cell clone expressing a useful antigenspecificity. In addition to circumventing the process of cloningvariable region genes, the level of expression of chimeric antibody ishigher when the gene is at its natural chromosomal location, rather thanat a random position in the genome. Detailed methods for preparation ofchimeric (humanized) antibodies can be found in U.S. Pat. No. 5,482,856.

In another embodiment, this invention provides for fully humanantibodies against selected Neisseria meningitidis ORF polypeptides.Human antibodies consist entirely of characteristically humanimmunoglobulin sequences. The human antibodies of this invention can beproduced in using a wide variety of methods (see, e.g., Larrick et al.,U.S. Pat. No. 5,001,065, for review).

In one preferred embodiment, the human antibodies of the presentinvention are produced initially in trioma cells. Genes encoding theantibodies are then cloned and expressed in other cells, such asnonhuman mammalian cells.

The general approach for producing human antibodies by trioma technologyis described by Ostberg et al. (1983), Hybridoma 2: 361-367, Ostberg,U.S. Pat. No. 4,634,664, and Engelman et al., U.S. Pat. No. 4,634,666.The antibody-producing cell lines obtained by this method are calledtriomas because they are descended from three cells; two human and onemouse. Triomas have been found to produce antibody more stably thanordinary hybridomas made from human cells.

Preparation of trioma cells requires an initial fusion of a mousemyeloma cell line with unimmortalized human peripheral B lymphocytes.This fusion generates a xenogeneic hybrid cell containing both human andmouse chromosomes (see, Engelman, supra.). Xenogeneic cells that havelost the capacity to secrete antibodies are selected. Preferably, axenogeneic cell is selected that is resistant to a selectable markersuch as 8-azaguanine. Cells possessing resistance to 8-azaguanine areunable to propagate on hypoxanthine-aminopterin-thymidine (HAT) orazaserine-hypoxanthine (AH) media.

The capacity to secrete antibodies is conferred by a further fusionbetween the xenogeneic cell and B-lymphocytes immunized against aselected Neisseria meningitidis ORF polypeptide, or an epitope thereof.The B-lymphocytes are obtained from the spleen, blood or lymph nodes ofhuman donor. If antibodies against a specific antigen rather than a fulllength polypeptide. Alternatively, B-lymphocytes are obtained from anunimmunized individual and stimulated with a polypeptide, or a epitopethereof, in vitro. In a further variation, B-lymphocytes are obtainedfrom an infected, or otherwise immunized individual, and thenhyperimmunized by exposure to a selected Neisseria meningitidis ORFpolypeptide for about seven to fourteen days, in vitro.

The immunized B-lymphocytes prepared by one of the above procedures arefused with a xeonogenic hybrid cell by well known methods. For example,the cells are treated with 40-50% polyethylene glycol of MW 1000-4000,at about 37° C. for about 5-10 min. Cells are separated from the fusionmixture and propagated in media selective for the desired hybrids. Whenthe xenogeneic hybrid cell is resistant to 8-azaguanine, immortalizedtrioma cells are conveniently selected by successive passage of cells onHAT or AH medium. Other selective procedures are, of course, possibledepending on the nature of the cells used in fusion. Clones secretingantibodies having the required binding specificity are identified byassaying the trioma culture medium for the ability to bind to a selectedNeisseria meningitidis polypeptide or an epitope thereof. Triomasproducing human antibodies having the desired specificity are subcloned,e.g., by the limiting dilution technique, and grown in vitro, in culturemedium, or are injected into selected host animals and grown in vivo.

The trioma cell lines obtained are then tested for the ability to bind apolypeptide or an epitope thereof. Antibodies are separated from theresulting culture medium or body fluids by conventionalantibody-fractionation procedures, such as ammonium sulfateprecipitation, DEAE cellulose chromatography and affinitychromatography.

Although triomas are genetically stable they do not produce antibodiesat very high levels. Expression levels can be increased by cloningantibody genes from the trioma into one or more expression vectors, andtransforming the vector into a cell line such as the cell linestypically used for expression of recombinant or humanizedimmunoglobulins. As well as increasing yield of antibody, this strategyoffers the additional advantage that immunoglobulins are obtained from acell line that does not have a human component, and does not thereforeneed to be subjected to the extensive viral screening required for humancell lines.

The genes encoding the heavy and light chains of immunoglobulinssecreted by trioma cell lines are cloned according to methods, includingthe polymerase chain reaction, known in the art (see, e.g., Sambrook,and Berger & Kimmel, both supra). For example, genes encoding heavy andlight chains are cloned from a trioma's genomic DNA or cDNA produced byreverse transcription of the trioma's RNA. Cloning is accomplished byconventional techniques including the use of PCR primers that hybridizeto the sequences flanking or overlapping the genes, or segments ofgenes, to be cloned.

Typically, recombinant constructs comprise DNA segments encoding acomplete human immunoglobulin heavy chain and/or a complete humanimmunoglobulin light chain of an immunoglobulin expressed by a triomacell line. Alternatively, DNA segments encoding only a portion of theprimary antibody genes are produced, which portions possess bindingand/or effector activities. Other recombinant constructs containsegments of trioma cell line immunoglobulin genes fused to segments ofother immunoglobulin genes, particularly segments of other humanconstant region sequences (heavy and/or light chain). Human constantregion sequences can be selected from various reference sources,including but not limited to those listed in Kabat et al. (1987),Sequences of Proteins of Immunological Interest, U.S. Department ofHealth and Human Services.

In addition to the DNA segments encoding anti-ORF immunoglobulins orfragments thereof, other substantially homologous modifiedimmunoglobulins can be readily designed and manufactured utilizingvarious recombinant DNA techniques known to those skilled in the artsuch as site-directed mutagenesis (see Gillman & Smith (1979) Gene, 8:81-97; Roberts et al. (1987) Nature, 328: 731-734). Such modifiedsegments will usually retain antigen binding capacity and/or effectorfunction. Moreover, the modified segments are usually not so far changedfrom the original trioma genomic sequences to prevent hybridization tothese sequences under stringent conditions. Because, like many genes,immunoglobulin genes contain separate functional regions, each havingone or more distinct biological activities, the genes may be fused tofunctional regions from other genes to produce fusion proteins (e.g.,immunotoxins) having novel properties or novel combinations orproperties.

The recombinant polynucleotide constructs will typically include anexpression control sequence operably linked to the coding sequences,including naturally-associated or heterologous promoter regions.Preferably, the expression control sequences will be eukaryotic promotersystems in vectors capable of transforming or transfecting eukaryotichost cells. Once the vector has been incorporated into the appropriatehost, the host is maintained under conditions suitable for high levelexpression of the nucleotide sequences, and the collection andpurification of the human immunoglobulins.

These expression vectors are typically replicable in the host organismseither as episomes or as an integral part of the host chromosomal DNA.Commonly, expression vectors will contain selection markers, e.g.,ampicillin-resistance or hygromycin-resistance, to permit detection ofthose cells transformed with the desired DNA sequences. In general,prokaryotes or eukaryotic cells are used for cloning the DNA sequencesencoding a human immunoglobulin chain.

Other approaches include in vitro immunization of human blood. In thisapproach, human blood lymphocytes capable of producing human antibodiesare produced. Human peripheral blood is collected from the patient andis treated to recover mononuclear cells. The suppressor T-cells then areremoved and remaining cells are suspended in a tissue culture medium towhich is added the antigen and autologous serum and, preferably, anonspecific lymphocyte activator. The cells then are incubated for aperiod of time so that they produce the specific antibody desired. Thecells then can be fused to human myeloma cells to immortalize the cellline, thereby to permit continuous production of antibody (see U.S. Pat.No. 4,716,111).

In another approach, mouse-human hybridomas which produce humanantibodies are prepared (see, e.g., 5,506,132). Other approaches includeimmunization of mice transformed to express human immunoglobulin genes,and phage display screening (Vaughan et al. supra.).

Cell-Mediated Immune Responses

In addition to the production of antibodies, the present inventionprovides for cell-mediated immune responses against Neisseriameningitidis. As above, a polypeptide of the invention (e.g., ORF 1 (SEQID NO:2), ORF 2a (SEQ ID NO:4), ORF 2b (SEQ ID NO:5), or ORF 3 (SEQ IDNO:7), or a subsequence thereof) is administered to a mammal. Theproliferation effect of these antigens is tested in a standard MLRassay. MLR assays or “mixed lymphocyte response” assays are the standardin vitro assay of antigen presenting function in cellular immunity. Theassay measures the proliferation of T cells after stimulation by aselected antigen-presenting cell type. The number of T cells producedare typically characterized by measuring T cell proliferation based onincorporation of ³H-thymidine in culture. Similar methods are used invivo in nude of SCID mouse models. See also, Paul (supra) at chapter 31.The most commonly measured from of cell-mediated immune response is acytotoxic T-lymphocyte (CTL) response.

Antigenic peptides are used to elicit CTL ex vivo. The resulting CTL,can be used to treat chronic infections in patients that do not respondto other conventional forms of therapy, or will not respond to a peptidevaccine approach of therapy. Ex vivo CTL responses to a particularpathogen (infectious agent or tumor antigen) are induced by incubatingin tissue culture the patient's CTL precursor cells (CTLp) together witha source of antigen-presenting cells (APC) and the appropriateimmunogenic peptide. After an appropriate incubation time (typically 1-4weeks), in which the CTLp are activated and mature and expand intoeffector CTLs, the cells are infused back into the patient, where theywill destroy their specific target cell (e.g., an infected cell).

Detection of Neisseria meningitidis

As indicated above, Neisseria meningitidis infection causes serioushealth problems, and has the potential to reach epidemic proportions insome populations. Accordingly, new methods of detecting infection ofpatients by Neisseria meningitidis is of considerable value.

Thus, it is desirable to determine the presence or absence of Neisseriameningitidis in a patient, or to quantify the severity of infection, orquantify the expression of Neisseria meningitidis polypeptides ornucleic acids. In addition, the polypeptides of the invention are usedto detect antisera against the polypeptides, e.g., in patientspreviously infected with Neisseria meningitidis.

Detection of Neisseria meningitidis or antisera against Neisseriameningitidis is accomplished by assaying the products of the Neisseriameningitidis nucleic acids of the invention; the nucleic acidsthemselves, or antibodies against polypeptides encoded by the nucleicacids. It is desirable to determine whether polypeptide expression ispresent, absent, or abnormal (e.g. because of an abnormal gene productor because of abnormal expression).

The selected Neisseria meningitidis nucleic acid or nucleic acid product(i.e., an mRNA or polypeptide) is preferably detected and/or quantifiedin a biological sample. Such samples include, but are not limited to,cerebrospinal fluid, sputum, amniotic fluid, blood, blood cells (e.g.,white cells), tissue or fine needle biopsy samples, urine, peritonealfluid, and pleural fluid, or cells therefrom. Biological samples mayalso include sections of tissues such as frozen sections taken forhistological purposes. Although the sample is typically taken from ahuman patient, the assays can be used to detect Neisseria meningitidisor Neisseria meningitidis gene products in samples from any mammal, suchas dogs, cats, sheep, cattle, rodents, primates and pigs.

The sample may be pretreated as necessary by dilution in an appropriatebuffer solution or concentrated, if desired. Any of a number of standardaqueous buffer solutions, employing one of a variety of buffers, such asphosphate, Tris, or the like, at physiological pH can be used.

In one embodiment, this invention provides for methods of detectingand/or quantifying Neisseria meningitidis gene expression by assayingthe underlying gene (or a fragment thereof) or by assaying the genetranscript (mRNA). The assay can be for the presence or absence of thenormal gene or gene product, for the presence or absence of an abnormalgene or gene product, or quantification of the transcription levels ofnormal or abnormal gene products.

In a preferred embodiment, nucleic acid assays are performed with asample of nucleic acid isolated from the organism to be tested. In thesimplest embodiment, such a nucleic acid sample is the total mRNAisolated from a biological sample. The nucleic acid (e.g., eithergenomic DNA or mRNA) may be isolated from the sample according to any ofa number of methods well known to those of skill in the art.

Methods of isolating total DNA or mRNA are well known to those of skillin the art. For example, methods of isolation and purification ofnucleic acids are described in detail in Chapter 3 of LaboratoryTechniques in Biochemistry and Molecular Biology: Hybridization WithNucleic Acid Probes, Part I. Theory and Nucleic Acid Preparation, P.Tijssen, ed. Elsevier, N.Y. (1993) and Chapter 3 of LaboratoryTechniques in Biochemistry and Molecular Biology: Hybridization WithNucleic Acid Probes, Part I. Theory and Nucleic Acid Preparation, P.Tijssen, ed. Elsevier, N.Y. (1993)).

Frequently, it is desirable to amplify the nucleic acid sample prior tohybridization. Methods of “quantitative” amplification are well known tothose of skill in the art. For example, quantitative PCR involvessimultaneously co-amplifying a known quantity of a control sequenceusing the same primers. This provides an internal standard that may beused to calibrate the PCR reaction. Detailed protocols for quantitativePCR are provided in PCR Protocols, A Guide to Methods and Applications,Innis et al., Academic Press, Inc. N.Y., (1990). Other suitableamplification methods include, but are not limited to those describedsupra.

Amplification-based assays are well known to those of skill in the art(see, e.g., Innis supra.). The Neisseria meningitidis nucleic acidsequences provided are sufficient to teach one of skill to routinelyselect primers to amplify any portion of the gene. It is expected thatone of skill is thoroughly familiar with the theory and practice ofnucleic acid hybridization and primer selection. Gait, ed.Oligonucleotide Synthesis: A Practical Approach, IRL Press, Oxford(1984); W. H. A. Kuijpers Nucleic Acids Research 18(17), 5197 (1994); K.L. Dueholm J. Org. Chem. 59, 5767-5773 (1994); S. Agrawal (ed.) Methodsin Molecular Biology, volume 20; and Tijssen (1993) LaboratoryTechniques in biochemistry and molecular biology—hybridization withnucleic acid probes, e.g., part I chapter 2 “overview of principles ofhybridization and the strategy of nucleic acid probe assays”, Elsevier,N.Y. provide a basic guide to nucleic acid hybridization. Innis supraprovides an overview of primer selection. In addition, PCR amplificationproducts are optionally detected on a polymer array as described inFodor et al. (1991) Science, 251: 767-777; Sheldon et al. (1993)Clinical Chemistry 39(4): 718-719, and Kozal et al. (1996) NatureMedicine 2(7): 753-759.

Most typically, amplification primers are between 8 and 100 nucleotidesin length, and preferably between about 10 and 30 nucleotides in length.More typically, the primers are between about 15 and 25 nucleic acids inlength.

One of skill will recognize that the 3′ end of an amplification primeris more important for PCR than the 5′ end. Investigators have reportedPCR products where only a few nucleotides at the 3′ end of anamplification primer were complementary to a DNA to be amplified. Inthis regard, nucleotides at the 5′ end of a primer can incorporatestructural features unrelated to the target nucleic acid; for instance,in one preferred embodiment, a sequencing primer hybridization site (ora complement to such as primer, depending on the application) isincorporated into the amplification primer, where the sequencing primeris derived from a primer used in a standard sequencing kit, such as oneusing a biotinylated or dye-labeled universal M13 or SP6 primer.Alternatively, the primers optionally incorporate restrictionendonuclease sites. The primers are selected so that there is nocomplementarity between any known sequence which is likely to occur inthe sample to be amplified and any constant primer region. One of skillwill appreciate that constant regions in the primer sequences areoptional.

Typically, all primer sequences are selected to hybridize only to aperfectly complementary DNA, with the nearest mismatch hybridizationpossibility from known DNA sequences which are likely to occur in thesample to be amplified having at least about 50 to 70% hybridizationmismatches, and preferably 100% mismatches for the terminal 5nucleotides at the 3′ end of the primer.

The primers are selected so that no secondary structure forms within theprimer. Self-complementary primers have poor hybridization properties,because the complementary portions of the primers self hybridize (i.e.,form hairpin structures). The primers are also selected so that theprimers do not hybridize to each other, thereby preventing duplexformation of the primers in solution, and possible concatenation of theprimers during PCR. If there is more than one constant region in theprimer, the constant regions of the primer are selected so that they donot self-hybridize or form hairpin structures.

Where sets of amplification primers (i.e., the 5′ and 3′ primers usedfor exponential amplification) are of a single length, the primers areselected so that they have roughly the same, and preferably exactly thesame overall base composition (i.e., the same A+T to G+C ratio ofnucleic acids). Where the primers are of differing lengths, the A+T toG+C ratio is determined by selecting a thermal melting temperature forthe primer-DNA hybridization, and selecting an A+T to G+C ratio andprobe length for each primer which has approximately the selectedthermal melting temperature.

One of skill will recognize that there are a variety of possible ways ofperforming the above selection steps, and that variations on the stepsare appropriate. Most typically, selection steps are performed usingsimple computer programs to perform the selection as outlined above;however, all of the steps are optionally performed manually. Oneavailable computer program for primer selection is the MacVector programfrom Kodak. In addition to commercially available programs for primerselection, one of skill can easily design simple programs for any of thepreferred selection steps. Amplification primers can be selected toprovide amplification products that span specific deletions,truncations, and insertions in an amplification target, therebyfacilitating the detection of specific abnormalities such as atransposon insertion as described herein.

Where it is desired to quantify the transcription level (and therebyexpression) of a normal or mutated Neisseria meningitidis gene is asample, the nucleic acid sample is one in which the concentration of themRNA transcript(s) of the gene, or the concentration of the nucleicacids derived from the mRNA transcript(s), is proportional to thetranscription level (and therefore expression level) of that gene.Similarly, it is preferred that the hybridization signal intensity beproportional to the amount of hybridized nucleic acid. While it ispreferred that the proportionality be relatively strict (e.g., adoubling in transcription rate results in a doubling in mRNA transcriptin the sample nucleic acid pool and a doubling in hybridization signal),one of skill will appreciate that the proportionality can be morerelaxed and even non-linear. Thus, for example, an assay where a 5 folddifference in concentration of a target mRNA results in a 3 to 6 folddifference in hybridization intensity is sufficient for most purposes.Where more precise quantification is required appropriate controls canbe run to correct for variations introduced in sample preparation andhybridization as described herein. In addition, serial dilutions of“standard” target mRNAs can be used to prepare calibration curvesaccording to methods well known to those of skill in the art. Of course,where simple detection of the presence or absence of a transcript isdesired, no elaborate control or calibration is required.

Neisseria meningitidis polypeptide assays.

The expression of selected Neisseria meningitidis polypeptides can alsobe detected and/or quantified by detecting or quantifying the expressedpolypeptide. The polypeptides can be detected and quantified by any of anumber of means well known to those of skill in the art. These mayinclude analytic biochemical methods such as electrophoresis, capillaryelectrophoresis, high performance liquid chromatography (HPLC), thinlayer chromatography (TLC), hyperdiffusion chromatography, and the like,or various immunological methods such as fluid or gel precipitinreactions, immunodiffusion (single or double), immunoelectrophoresis,radioimmunoassay(RIA), enzyme-linked immunosorbent assays (ELISAs),immunofluorescent assays, western blotting, and the like.

In a particularly preferred embodiment, the polypeptides are detected inan electrophoretic protein separation, more preferably in atwo-dimensional electrophoresis, while in a most preferred embodiment,the polypeptides are detected using an immunoassay.

As used herein, an immunoassay is an assay that utilizes an antibody tospecifically bind to the analyte (e.g., selected polypeptide, such asORF 1 (SEQ ID NO:2), ORF 2a (SEQ ID NO:4), ORF 2b (SEQ ID NO:5), or ORF3 (SEQ ID NO:7)). The immunoassay is thus characterized by detection ofspecific binding of a polypeptide to an anti-polypeptide antibody, asopposed to the use of other physical or chemical properties to isolate,target, and quantify the analyte.

As indicated above, the presence or absence of polypeptides in abiological sample can be determined using electrophoretic methods. Meansof detecting proteins using electrophoretic techniques are well known tothose of skill in the art (see generally, R. Scopes (1982) ProteinPurification, Springer-Verlag, N.Y.; Deutscher, (1990) Methods inEnzymology Vol. 182: Guide to Protein Purification., Academic Press,Inc., N.Y.).

In a preferred embodiment, the polypeptides are detected and/orquantified using any of a number of well recognized immunologicalbinding assays (see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110;4,517,288; and 4,837,168). For a review of the general immunoassays, seealso Methods in Cell Biology Volume 37: Antibodies in Cell Biology,Asai, ed. Academic Press, Inc. New York (1993); Basic and ClinicalImmunology 7th Edition, Stites & Terr, eds. (1991). Immunologicalbinding assays (or immunoassays) typically utilize a “capture agent” tospecifically bind to and often immobilize the analyte. The capture agentis a moiety that specifically binds to the analyte. In a preferredembodiment, the capture agent is an antibody that specifically bindspolypeptide(s) or polypeptide subsequences (e.g., antigenic domainswhich specifically bind to the antibody). In a second preferredembodiment, the capture agent is the polypeptide and the analyte isantisera comprising an antibody which specifically binds to thepolypeptide.

Immunoassays often utilize a labeling agent to specifically bind to andlabel the binding complex formed by the capture agent and the analyte.The labeling agent may itself be one of the moieties comprising theantibody/analyte complex. Thus, the labeling agent may be a labeledpolypeptide or a labeled anti-polypeptide antibody. Alternatively, thelabeling agent may be a third moiety, such as another antibody, thatspecifically binds to the antibody/polypeptide complex.

In a preferred embodiment, the labeling agent is a second antibodybearing a label. Alternatively, the second antibody may lack a label,but it may, in turn, be bound by a labeled third antibody specific toantibodies of the species from which the second antibody is derived. Thesecond antibody can be modified with a detectable moiety, such asbiotin, to which a third labeled molecule can specifically bind, such asenzyme-labeled streptavidin.

Other proteins capable of specifically binding immunoglobulin constantregions, such as streptococcal protein A or protein G may also be usedas the label agent. These proteins are normal constituents of the cellwalls of streptococcal bacteria. They exhibit a strong non-immunogenicreactivity with immunoglobulin constant regions from a variety ofspecies (see, generally Kronval, et al. (1973) J. Immunol., 111:1401-1406, and Akerstrom, et al. (1985) J. Immunol., 135: 2589-2542).

Throughout the assays, incubation and/or washing steps are optionallyperformed after each combination of reagents. Incubation steps can varyfrom about 5 seconds to several hours, preferably from about 5 minutesto about 24 hours. However, the incubation time will depend upon theassay format, analyte, volume of solution, concentrations, and the like.Usually, the assays will be carried out at ambient temperature, althoughthey can be conducted over a range of temperatures, such as 10° C. to40° C.

Immunoassays for detecting polypeptides may be either competitive ornoncompetitive. Noncompetitive immunoassays are assays in which theamount of captured analyte is directly measured. In one preferred“sandwich” assay, for example, the capture agent can be bound directlyto a solid substrate where they are immobilized. These immobilizedcapture agent then captures analyte present in the test sample. Theanalyte thus immobilized is then bound by a labeling agent, such as asecond antibody bearing a label. Alternatively, the second antibody maylack a label, but it may, in turn, be bound by a labeled third antibodyspecific to antibodies of the species from which the second antibody isderived. The second can be modified with a detectable moiety, such asbiotin, to which a third labeled molecule can specifically bind, such asenzyme-labeled streptavidin.

In competitive assays, the initial amount of analyte present in thesample is measured indirectly by measuring the amount of an added(exogenous) analyte displaced (or competed away) from a capture agent bythe analyte present in the sample. In one competitive assay, a knownamount of, in this case, analyte is added to the sample and the sampleis then contacted with a capture agent. The amount of exogenous analytebound to the capture agent is inversely proportional to the initialanalyte present in the sample.

In a preferred embodiment, western blot (immunoblot) analysis is used todetect and quantify the presence of selected Neisseria meningitidis inthe sample. The technique generally comprises separating sample proteinsby gel electrophoresis on the basis of molecular weight, transferringthe separated proteins to a suitable solid support (such as anitrocellulose filter, a nylon filter, or derivatized nylon filter), andincubating the sample with the antibodies that specifically bind theselected polypeptide. The antibodies specifically bind to polypeptide onthe solid support. These antibodies are optionally directly labeled oralternatively are optionally subsequently detected using labeledantibodies (e.g., labeled sheep anti-mouse antibodies) that specificallybind to the selected polypeptide.

Other assay formats include liposome immunoassays (LIA), which useliposomes designed to bind specific molecules (e.g., antibodies) andrelease encapsulated reagents or markers. The released chemicals arethen detected according to standard techniques (see, Monroe et al.(1986) Amer. Clin. Prod. Rev. 5:34-41). Enzyme linked assays (e.g.,ELISA assays) are also preferred.

The assays of this invention as scored (as positive or negative forNeisseria meningitidis or a selected Neisseria meningitidis polypeptide)according to standard methods well known to those of skill in the art.The particular method of scoring will depend on the assay format andchoice of label. For example, a western blot assay can be scored byvisualizing the colored product produced by the enzymatic label. Aclearly visible colored band or spot at the correct molecular weight isscored as a positive result, while the absence of a clearly visible spotor band is scored as a negative. In a preferred embodiment, a positivetest will show a signal intensity (e.g., polypeptide quantity) at leasttwice that of the background and/or control and more preferably at least3 times or even at least 5 times greater than the background and/ornegative control.

One of skill in the art will appreciate that it is often desirable toreduce non-specific binding in immunoassays. Particularly, where theassay involves an antigen or antibody immobilized on a solid substrateit is desirable to minimize the amount of non-specific binding to thesubstrate. Means of reducing such non-specific binding are well known tothose of skill in the art. Typically, this involves coating thesubstrate with a proteinaceous composition. In particular, proteincompositions such as bovine serum albumin (BSA), nonfat powdered milk,and gelatin.

The particular label or detectable group used in the assay is not acritical aspect of the invention, so long as it does not significantlyinterfere with the specific binding of the antibody used in the assay.The detectable group can be any material having a detectable physical orchemical property. Such detectable labels have been well-developed inthe field of immunoassays and, in general, most any label useful in suchmethods can be applied to the present invention. Thus, a label is anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful labels inthe present invention include magnetic beads (e.g. Dynabeads™),fluorescent dyes (e.g., fluorescein isothiocyanate, texas red,rhodamine, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase andothers commonly used in an ELISA), and colorimetric labels such ascolloidal gold or colored glass or plastic (e.g. polystyrene,polypropylene, latex, etc.) beads.

The label may be coupled directly or indirectly to the desired componentof the assay according to methods well known in the art. As indicatedabove, a wide variety of labels may be used, with the choice of labeldepending on sensitivity required, ease of conjugation with thecompound, stability requirements, available instrumentation, anddisposal provisions.

Non-radioactive labels are often attached by indirect means. Generally,a ligand molecule (e.g., biotin) is covalently bound to the molecule.The ligand then binds to an anti-ligand (e.g., streptavidin) moleculewhich is either inherently detectable or covalently bound to a signalsystem, such as a detectable enzyme, a fluorescent compound, or achemiluminescent compound. A number of ligands and anti-ligands can beused. Where a ligand has a natural anti-ligand, for example, biotin,thyroxine, and cortisol, it can be used in conjunction with the labeled,naturally occurring anti-ligands. Alternatively, any haptenic orantigenic compound can be used in combination with an antibody.

The molecules can also be conjugated directly to signal generatingcompounds, e.g., by conjugation with an enzyme or fluorophore. Enzymesof interest as labels will primarily be hydrolases, particularlyphosphatases, esterases and glycosidases, or oxidoreductases,particularly peroxidases. Fluorescent compounds include fluorescein andits derivatives, rhodamine and its derivatives, dansyl, umbelliferone,etc. Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal producing systems which may be used, see, U.S. Pat.No. 4,391,904).

Means of detecting labels are well known to those of skill in the art.Thus, for example, where the label is a radioactive label, means fordetection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally simple colorimetriclabels may be detected simply by observing the color associated with thelabel. Thus, in various dipstick assays, conjugated gold often appearspink, while various conjugated beads appear the color of the bead.

Some assay formats do not require the use of labeled components. Forinstance, agglutination assays can be used to detect the presence of thetarget antibodies. In this case, antigen-coated particles areagglutinated by samples comprising the target antibodies. In thisformat, none of the components need be labeled and the presence of thetarget antibody is detected by simple visual inspection.

As mentioned above, depending upon the assay, various components,including the antigen, target antibody, or anti-antibody, may be boundto a solid surface. Many methods for immobilizing biomolecules to avariety of solid surfaces are known in the art. For instance, the solidsurface may be a membrane (e.g., nitrocellulose), a microtiter dish(e.g., PVC, polypropylene, or polystyrene), a test tube (glass orplastic), a dipstick (e.g. glass, PVC, polypropylene, polystyrene,latex, and the like), a microcentrifuge tube, or a glass or plasticbead. The desired component may be covalently bound or noncovalentlyattached through nonspecific bonding.

A wide variety of organic and inorganic polymers, both natural andsynthetic may be employed as the material for the solid surface.Illustrative polymers include polyethylene, polypropylene,poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethyleneeterphthalate), rayon, nylon, poly(vinyl butyrate), polyvinylidenedifluoride (PVDF), silicones, polyformaldehyde, cellulose, celluloseacetate, nitrocellulose, and the like. Other materials which may beemployed, include paper, glasses, ceramics, metals, metalloids,semiconductive materials, cements or the like. In addition, are includedsubstances that form gels, such as proteins (e.g., gelatins),lipopolysaccharides, silicates, agarose and polyacrylamides can be used.Polymers which form several aqueous phases, such as dextrans,polyalkylene glycols or surfactants, such as phospholipids, long chain(12-24 carbon atoms) alkyl ammonium salts and the like are alsosuitable. Where the solid surface is porous, various pore sizes may beemployed depending upon the nature of the system.

In preparing the surface, a plurality of different materials may beemployed, particularly as laminates, to obtain various properties. Forexample, protein coatings, such as gelatin can be used to avoidnon-specific binding, simplify covalent conjugation, enhance signaldetection or the like.

If covalent bonding between a compound and the surface is desired, thesurface will usually be polyfunctional or be capable of beingpolyfunctionalized. Functional groups which may be present on thesurface and used for linking can include carboxylic acids, aldehydes,amino groups, cyano groups, ethylenic groups, hydroxyl groups, mercaptogroups and the like. The manner of linking a wide variety of compoundsto various surfaces is well known and is amply illustrated in theliterature. See, for example, Immobilized Enzymes, Ichiro Chibata,Halsted Press, New York, 1978, and Cuatrecasas (1970) J. Biol. Chem. 2453059).

In addition to covalent bonding, various methods for noncovalentlybinding an assay component can be used. Noncovalent binding is typicallynonspecific absorption of a compound to the surface. Typically, thesurface is blocked with a second compound to prevent nonspecific bindingof labeled assay components. Alternatively, the surface is designed suchthat it nonspecifically binds one component but does not significantlybind another. For example, a surface bearing a lectin such asConcanavalin A will bind a carbohydrate containing compound but not alabeled protein that lacks glycosylation. Various solid surfaces for usein noncovalent attachment of assay components are reviewed in U.S. Pat.Nos. 4,447,576 and 4,254,082.

Detection kits

The present invention also provides kits for the diagnosis of patientsinfected with Neisseria meningitidis. The kits preferably include one ormore reagents for determining the presence or absence of a selectedNeisseria meningitidis nucleic acid or protein, i.e., any of the nucleicacids or proteins described herein. Preferred reagents include nucleicacid probes that specifically bind to Seq 1 (SEQ ID NO:1), Seq 2 (SEQ IDNO: 3), Seq 3 (SEQ ID NO:6), or Seq 4 (SEQ ID NO:8) cDNA correspondingto Seq 1 (SEQ ID NO:1), Seq 2 (SEQ ID NO:3), Seq 3 (SEQ ID NO:6), or Seq4 (SEQ ID NO:8), or a subsequence thereof; probes that specifically bindto an abnormal Neisseria meningitidis gene (e.g., one containingpremature truncations, insertions, or deletions), and antibodies thatspecifically bind to polypeptides or subsequences thereof. The antibodyor hybridization probe may be free or immobilized on a solid supportsuch as a test tube, a microtiter plate, a dipstick or the like. The kitmay also contain instructional materials teaching the use of theantibody or hybridization probe in an assay for the detection ofNeisseria meningitidis, a container or other packaging material or thelike.

The kits may include alternatively, or in combination with any of theother components described herein, an antibody which specifically bindsa polypeptide of the invention. The antibody can be monoclonal orpolyclonal. The antibody can be conjugated to another moiety such as alabel and/or it can be immobilized on a solid support (substrate).

The kits also optionally include a second antibody for detection ofpolypeptide/antibody complexes or for detection of hybridized nucleicacid probes. The kits optionally include appropriate reagents fordetection of labels, positive and negative controls, washing solutions,dilution buffers and the like.

Intracellular Immunization and Gene Therapy

In one preferred class of embodiments, the nucleic acids of theinvention are used in cell transformation procedures for intracellularimmunization and gene therapy to inhibit or prevent meningitis caused byNeisseria meningitidis serogroup B. Gene therapy provides methods forcombating chronic infectious diseases. In vitro, ex vivo and in vivoprocedures are used. The nucleic acids of the invention optionallyencode antisense oligonucleotides which bind to selected Neisseriameningitidis nucleic acids (e.g., RNAs encoded by Seq 1 (SEQ ID NO:1),Seq 2 (SEQ ID NO:3), Seq 3 (SEQ ID NO:6), or Seq 4 (SEQ ID NO:8); seeFIGS. 5, 6, 7 and 4, respectively) with high affinity. Theseoligonucletides are typically cloned into gene therapy vectors that arecompetent to transform cells (such as human or other mammalian cells) invitro and/or in vivo.

Several approaches for introducing nucleic acids into cells in vivo, exvivo and in vitro have been used. These include liposome based genedelivery (Debs and Zhu (1993) WO 93/24640; Mannino and Gould-Fogerite(1988) BioTechniques 6(7): 682-691; Rose U.S. Pat. No. 5,279,833;Brigham (1991) WO 91/06309; and Felgner et al. (1987) Proc. Natl. Acad.Sci. USA 84, 7413—7414) and replication-defective retroviral vectorsharboring a therapeutic polynucleotide sequence as part of theretroviral genome (see, e.g., Miller et al. (1990) Mol. Cell. Biol.10:4239 (1990); Koiberg (1992) J. NIH Res. 4:43, and Cornetta et al.Hum. Gene Ther. 2:215 (1991)).

For a review of gene therapy procedures, see, Anderson, Science (1992)256:808-813; Nabel and Felgner (1993) TIBTECH 11:211-217; Mitani andCaskey (1993) TIBTECH 11:162-166; Mulligan (1993) Science 926-932;Dillon (1993) TIBTECH 11:167-175; Miller (1992) Nature 357:455-460; VanBrunt (1988) Biotechnology 6(10):1149-1154; Vigne (1995) RestorativeNeurology and Neuroscience 8:35-36; Kremer and Perricaudet (1995)British Medical Bulletin 51(1) 31-44; Haddada et al. (1995) in CurrentTopics in Microbiology and Immunology Doerfler and Böhm (eds)Springer-Verlag, Heidelberg Germany; and Yu et al., Gene Therapy (1994)1:13-26.

Widely used retroviral vectors include those based upon murine leukemiavirus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiencyvirus (SIV), human immuno deficiency virus (HIV), and combinationsthereof. See, e.g., Buchscher et al. (1992) J. Virol. 66(5)) 2731-2739;Johann et al. (1992) J. Virol. 66 (5):1635-1640 (1992); Sommerfelt etal., (1990) Virol. 176:58-59; Wilson et al. (1989) J. Virol.63:2374-2378; Miller et al., J. Virol. 65:2220-2224 (1991); Wong-Staalet al., PCT/US94/05700, and Rosenburg and Fauci (1993) in FundamentalImmunology, Third Edition Paul (ed) Raven Press, Ltd., New York and thereferences therein, and Yu et al., Gene Therapy (1994) supra). Thevectors are optionally psuedotyped to extend the host range of thevector to cells which are not infected by the retrovirus correspondingto the vector. The vesicular stomatitis virus envelope glycoprotein(VSV-G) has been used to construct VSV-G-pseudotyped HIV vectors whichcan infect hematopoietic stem cells (Naldini et al. (1996) Science272:263, and Akkina et al. (1996) J Virol 70:2581).

Adeno-associated virus (AAV)-based vectors are also used to transducecells with target nucleic acids, e.g., in the in vitro production ofnucleic acids and peptides, and in in vivo and ex vivo gene therapyprocedures. See, West et al. (1987) Virology 160:38-47; Carter et al.(1989) U.S. Pat. No. 4,797,368; Carter et al. WO 93/24641 (1993); Kotin(1994) Human Gene Therapy 5:793-801; Muzyczka (1994) J. Clin. Invst.94:1351 for an overview of AAV vectors. Construction of recombinant AAVvectors are described in a number of publications, including Lebkowski,U.S. Pat. No. 5,173,414; Tratschin et al. (1985) Mol. Cell. Biol.5(11):3251-3260; Tratschin, et al. (1984) Mol. Cell. Biol., 4:2072-2081;Hermonat and Muzyczka (1984) Proc. Natl. Acad. Sci. USA, 81:6466-6470:McLaughlin et al. (1988) and Samulski et al. (1989) J. Virol.,63:03822-3828. Cell lines that can be transformed by rAAV include thosedescribed in Lebkowski et al. (1988) Mol. Cell. Biol., 8:3988-3996.

Ex vivo methods for inhibiting Neisseria meningitidis replication in acell in an organism involve transducing the cell ex vivo with a nucleicacid of this invention which expresses an antisense oligonucleotide ofthe invention, and introducing the cell into the organism. The cultureof cells used in conjunction with the present invention, including celllines and cultured cells from tissue or blood samples is well known inthe art. Freshney (Culture of Animal Cells, a Manual of Basic Technique,third edition Wiley-Liss, New York (1994)) and the references citedtherein provides a general guide to the culture of cells. Transformedcells are cultured by means well known in the art. See, also Kuchler etal. (1977) Biochemical Methods in Cell Culture and Virology, Kuchler, R.J., Dowden, Hutchinson and Ross, Inc., and Atlas (1993) CRC Handbook ofMicrobiological Media (Parks ed) CRC press, Boca Raton, Fla. Mammaliancell systems often will be in the form of monolayers of cells, althoughmammalian cell suspensions are also used. Alternatively, cells can bederived from those stored in a cell bank (e.g., a blood bank).

In one preferred use of the invention, expression of an oligonucleotideinhibits Neisseria meningitidis replication in any of those cellsalready infected with Neisseria meningitidis, in addition to conferringa protective effect to cells which are not infected. Thus, an organisminfected with Neisseria meningitidis can be treated for the infection bytransducing a population of its cells with a vector of the invention andintroducing the transduced cells back into the organism. Thus, thepresent invention provides a method of protecting cells in vitro, exvivo or in vivo, even when the cells are already infected with the virusagainst which protection is sought.

A ribozyme is a catalytic antisense RNA molecule that cleaves other RNAmolecules having particular target nucleic acid sequences. Generalmethods for the construction of ribozymes against selected targets,including hairpin ribozymes, hammerhead ribozymes, RNAse P ribozymes(i.e., ribozymes derived from the naturally occurring RNAse P ribozymefrom prokaryotes or eukaryotes) are known in the art. Castanotto et al(1994) Advances in Pharmacology 25:289-317 provides and overview ofribozymes in general, including group I ribozymes, hammerhead ribozymes,hairpin ribozymes RNAse P, and axhead ribozymes.

Briefly, two types of ribozymes that are particularly useful in thisinvention include the hairpin ribozyme and the hammerhead ribozyme. Thehammerhead ribozyme (see, Rossie et al. (1991) Pharmac. Ther.50:245-254; Forster and Symons (1987) Cell 48:211-220; Haseloff andGerlach (1988) Nature 328:596-600; Walbot and Bruening (1988) Nature334:196; Haseloff and Gerlach (1988) Nature 334:585; and Dropulic et aland Castanotto et al., and the references cited therein, supra) and thehairpin ribozyme (see, e.g., Hampel et al. (1990) Nucl. Acids Res.18:299-304; Hempel et al., (1990) European Patent Publication No. 0 360257; U.S. Pat. No. 5,254,678; Wong-Staal et al., PCT/US94/05700; Ojwanget al. (1993) Proc Natl Acad Sci USA 90:6340-6344; Yamada et al. (1994)Human Gene Therapy 1:39-45; Leavitt et al. (1995) Proc Natl Acad Sci USA92:699-703; Leavitt et al. (1994) Human Gene Therapy 5:1151-1120; andYamada et al. (1994) Virology 205:121-126) are catalytic moleculeshaving antisense and endoribonucleotidase activity.

The typical sequence requirement for the GUC hairpin ribozyme is a RNAsequence consisting of NNNG/CN*GUCNNNNNNNN (SEQ ID NO:9) (where N*G isthe cleavage site, and where N is any of G, U, C, or A). The sequencerequirement at the cleavage site for the hammerhead ribozyme is any RNAsequence consisting of NUX (where N is any of G, U, C, or A and Xrepresents C, U or A). Accordingly, the same target within the hairpinleader sequence, GUC, is targetable by the hammerhead ribozyme. Theadditional nucleotides of the hammerhead ribozyme or hairpin ribozyme isdetermined by the common target flanking nucleotides and, e.g., thehammerhead consensus sequences.

Altman (1995) Biotechnology 13:327-329 and the references thereindescribe the use of RNAse P as a therapeutic agent directed against fluvirus. Similar therapeutic approaches can be used against selectedNeisseria meningitidis RNAs by incorporating RNAse P catalytic domainsinto the antisense molecules of the invention.

The anti sense molecules, including the ribozymes of this invention andDNA encoding the ribozymes, can be chemically synthesized as describedsupra, or prepared from a DNA molecule (that upon transcription yieldsan RNA molecule) operably linked to an appropriate promoter.

Reporter genes, Sites of Replication and Selectable Markers

To monitor the progress of cellular transduction, a marker or “reporter”gene is optionally encoded by the nucleic acids of the invention. Theinclusion of detectable markers provides a means of monitoring theinfection and stable transduction of target cells. Markers includecomponents of the beta-galactosidase gene, the firefly luciferase geneand the green fluorescence protein (see, e.g., Chalfie et al. (1994)Science 263:802).

The vectors of the invention optionally include features whichfacilitate the replication in more than one cell type. For example, thereplication of a plasmid as an episomal nucleic acid in mammalian cellscan be controlled by the large T antigen in conjunction with anappropriate origin of replication, such as the origin of replicationderived from the BK papovavirus. Many other features which permit avector to be grown in multiple cell types (e.g., shuttle vectors whichare replicated in prokaryotic and eukaryotic cells) are known.

Selectable markers which facilitate cloning of the vectors of theinvention are optionally included. Sambrook and Ausbel, both supra,provide an overview of selectable markers.

The present invention provides nucleic acids for the transformation ofcells in vitro and in vivo. These nucleic acids are typically packagedin vector particles. The nucleic acids are transfected into cellsthrough the interaction of the vector particle surrounding the nucleicacid and the cellular receptor for the vector. For example, cells whichare transfected by HIV based vectors in vitro include CD4⁺cells,including T-cells such as Molt-{fraction (4/8)} cells, SupT1 cells, H9cells, C8166 cells and myelomonocytic (U937) cells, as well as primaryhuman lymphocytes, and primary human monocyte-macrophage cultures,peripheral blood dendritic cells, follicular dendritic cells, epidermalLangerhans cells, megakaryocytes, microglia, astrocytes,oligodendroglia, CD8⁺cells, retinal cells, renal epithelial cells,cervical cells, rectal mucosa, trophoblastic cells, and cardiac myocytes(see also, Rosenburg and Fauci Rosenburg and Fauci (1993) in FundamentalImmunology, Third Edition Paul (ed) Raven Press, Ltd., New York). AAVbased vectors transduce most mammalian cells. In one particularlypreferred class of embodiments, the nucleic acids of the invention areused in cell transformation procedures for gene therapy.

In addition to viral particles, a variety of protein coatings can beused to target nucleic acids to selected cell types.Transferrin-poly-cation conjugates enter cells which comprisetransferrin receptors, See, e.g., Zenke et al (1990) Proc. Natl. Acad.Sci. USA 87:3655-3659; Curiel (1991) Proc. Natl. Acad Sci USA88:8850-8854 and Wagner et al. (1993) Proc. Natl. Acad. Sci. USA89:6099-6013.

Naked plasmid DNA bound electrostatically to poly-l-lysine orpoly-l-lysine-transferrin which has been linked to defective adenovirusmutants can be delivered to cells with transfection efficienciesapproaching 90% (Curiel et al. (1991) Proc Natl Acad Sci USA88:8850-8854; Cotten et al. (1992) Proc Natl Acad Sci USA 89:6094-6098;Curiel et al. (1992) Hum Gene Ther 3:147-154; Wagner et al. (1992) ProcNatl Acad Sci USA 89:6099-6103; Michael et al. (1993) J Biol Chem268:6866-6869; Curiel et al. (1992) Am J Respir Cell Mol Biol 6:247-252,and Harris et al. (1993) Am J Respir Cell Mol Biol 9:441-447). Theadenovirus-poly-l-lysine-DNA conjugate binds to the normal adenovirusreceptor and is subsequently internalized by receptor-mediatedendocytosis. The adenovirus-poly-l-lysine-DNA conjugate binds to thenormal adenovirus receptor and is subsequently internalized byreceptor-mediated endocytosis. Similarly, other virus-poly-l-lysine-DNAconjugates bind the normal viral receptor and are subsequentlyinternalized by receptor-mediated endocytosis. Accordingly, a variety ofviral particles can be used to target vector nucleic acids to cells.

In addition to, or in place of receptor-ligand mediated transduction,the vector nucleic acids of the invention are optionally complexed withliposomes to aid in cellular transduction. Liposome based gene deliverysystems are described in Debs and Zhu (1993) WO 93/24640; Mannino andGould-Fogerite (1988) BioTechniques 6(7):682-691; Rose U.S. Pat. No.5,279,833; Brigham (1991) WO 91/06309; and Felgner et al. (1987) Proc.Natl. Acad. Sci. USA 84:7413-7414.

Ex Vivo Transduction of Cells

Ex vivo methods for inhibiting viral replication in a cell in anorganism involve transducing the cell ex vivo with a therapeutic nucleicacid of this invention, and introducing the cell into the organism. Thecells are typically isolated or cultured from a patient. Alternatively,the cells can be those stored in a cell bank (e.g., a blood bank).

In one class of embodiments, the vectors of the invention inhibitNeisseria meningitidis replication in cells already infected withNeisseria meningitidis, in addition to conferring a protective effect tocells which are not infected by Neisseria meningitidis. Thus, anorganism infected with Neisseria meningitidis can be treated for theinfection by transducing a population of its cells with a vectorencoding an antisense molecule against a selected Neisseria meningitidisRNA and introducing the transduced cells back into the patient asdescribed herein. Thus, the present invention provides compositions andmethods for protecting cells in culture, ex vivo and in a patient, evenwhen the cells are already infected with the Neisseria meningitidis.

The culture of cells used in conjunction with the present invention,including cell lines and cultured cells from tissue or blood samples iswell known in the art. Freshney (Culture of Animal Cells, a Manual ofBasic Technique, third edition Wiley-Liss, New York (1994)) and thereferences cited therein provides a general guide to the culture ofcells. Transduced cells are cultured by means well known in the art.See, also Kuchler et al. (1977) Biochemical Methods in Cell Culture andVirology, Kuchler, R. J., Dowden, Hutchinson and Ross, Inc. Mammaliancell systems often will be in the form of monolayers of cells, althoughmammalian cell suspensions are also used. Illustrative examples ofmammalian cell lines include the HEC-1-B cell line, VERO and Hela cells,Chinese hamster ovary (CHO) cell lines, W138, BHK, Cos-7 or MDCK celllines (see, e.g., Freshney, supra).

In one embodiment, CD34⁺stem cells are optionally used in ex-vivoprocedures for cell transduction and gene therapy. The advantage tousing stem cells is that they can be introduced into a mammal (such asthe donor of the cells) where they will engraft in the bone marrow anddifferentiate into many different immune cell types.

In humans, CD34⁺cells can be obtained from a variety of sourcesincluding cord blood, bone marrow, and mobilized peripheral blood.Purification of CD34⁺cells can be accomplished by antibody affinityprocedures. An affinity column isolation procedure for isolatingCD34⁺cells is described by Ho et al. (1995) Stem Cells 13 (suppl.3):100-105. See also, Brenner (1993) Journal of Hematotherapy 2:7-17. Yuet al. (1995) PNAS 92:699-703 describe a method of transducingCD34⁺cells from human fetal cord blood using retroviral vectors.

Rather than using stem cells, T cells or B cells are also used in someembodiments in ex vivo procedures. Several techniques are known forisolating T and B cells. The expression of surface markers facilitatesidentification and purification of such cells. Methods of identificationand isolation of cells include FACS, incubation in flasks with fixedantibodies which bind the particular cell type and panning with magneticbeads.

Administration of Nucleic Acids, Gene Therapy Vectors, ImmunogenicCompositions and Transduced Cells

Nucleic acids (typically DNA) encoding the polypeptides of the inventionare administered to patients to elicit an immune response against thepolypeptides which they encode. DNA administered for this purpose isreferred to as a “DNA vaccine.” Methods of making and administering DNAas vaccines are known, and described, e.g., in Wolff et al., Science247:1465-1468 (1990). The nucleic acids of the invention, includingantisense molecules, are also optionally administered to inhibitNeisseria meningitidis replication in cells transduced by the vectors,as described supra.

In another aspect, the present invention is directed to administrationof immunogenic compositions and vaccines which contain as an activeingredient an immunogenically effective amount of an immunogenic peptideas described herein. The peptide(s) may be introduced into a mammal,including a human. The peptide is optionally linked to a carrier, or ispresent as a homopolymer or heteropolymer of active peptide units.Polymerization of multiple units of the polypeptides of the inventionprovides the advantage of increased immunological reaction and, wheredifferent peptides are used to make up the polymer, the additionalability to induce antibodies and/or CTLs that react with differentantigenic determinants of the virus or tumor cells. Useful carriers arewell known in the art, and include, e.g., thyroglobulin, albumins suchas human serum albumin, tetanus toxoid, polyamino acids such aspoly(lysine: glutamic acid), influenza, hepatitis B virus core protein,hepatitis B virus recombinant vaccine and the like. The vaccines canalso contain a physiologically tolerable (acceptable) diluent orexcipient such as water, phosphate buffered saline, or saline. Thevaccines and immunogenic compositions of the invention further typicallyinclude an adjuvant. Adjuvants such as incomplete Freund's adjuvant,aluminum phosphate, aluminum hydroxide, or alum are materials well knownin the art. CTL responses can be primed by conjugating peptides of theinvention to lipids. Upon immunization with a peptide composition asdescribed herein, the immune system of the host responds to the vaccineby producing antibodies and CTLs specific for the desired antigen,making the host resistant to later infection by Neisseria meningitidis,or resistant to developing chronic infection. In addition to thepolypeptides herein, known Neisseria meningitidis immunogens areoptionally present in any immunogenic or vaccine composition, therebyproviding an immune response against the both peptides of the inventionand known polypeptides. For therapeutic or immunization purposes, thepeptides of the invention can also be expressed by attenuated viralhosts, such as vaccinia or fowlpox. This approach involves the use ofvaccinia virus as a vector to express nucleotide sequences that encodethe peptides of the invention. Upon introduction into an acutely orchronically infected host or into a non-infected host, the recombinantvaccinia virus expresses the immunogenic peptide, and thereby elicits ahost CTL response. Vaccinia vectors and methods useful in immunizationprotocols are described in, e.g., U.S. Pat. No. 4,722,848, incorporatedherein by reference. Another vector is BCG (Bacille Calmette Guerin).BCG vectors are described in Stover et al. (1991) Nature 351:456-460. Awide variety of other vectors useful for therapeutic administration orimmunization with the peptides of the invention, e.g., Salmonella typhivectors and the like, will be apparent to those skilled in the art fromthe description herein.

Accordingly, the present invention provides for administration ofnucleic acids (e.g., DNA vaccines or cell transformation vectors),polypeptides, immunogenic compositions comprising a polypeptide, vaccinecomponents, and transduced cells (e.g., those made in ex vivo genetherapy or CTL procedures). Administration is by any of the routesnormally used for introducing a molecule into ultimate contact withblood or tissue cells. Administration is made in any suitable manner,preferably with pharmaceutically acceptable carriers. Suitable methodsof administering nucleic acids, proteins, vaccines, cells andimmunogenic compositions in the context of the present invention to apatient are available. Intra-muscular and subcutaneous administration isappropriate for, e.g., vaccines, DNA vaccines, and immunogeniccompositions. Parenteral administration such as intravenousadministration is a suitable method of administration for transducedcells and cell transformation vectors. Formulations of compositions tobe administered can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials.

Pharmaceutically acceptable excipients are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions of thepresent invention. Formulations suitable for parenteral administration,such as, for example, by intraarticular (in the joints), intravenous,intramuscular, intradermal, intraperitoneal, and subcutaneous routes,include aqueous and non-aqueous, isotonic sterile injection solutions,which can contain antioxidants, buffers, bacteriostats, and solutes thatrender the formulation isotonic with the blood of the intendedrecipient, and aqueous and non-aqueous sterile suspensions that caninclude suspending agents, solubilizers, thickening agents, stabilizers,and preservatives.

The dose administered to a patient, in the context of the presentinvention should be sufficient to effect a beneficial therapeuticresponse in the patient over time, such as a reduction in the level ofNeisseria meningitidis, or to inhibit infection by Neisseriameningitidis. The dose will be determined by the efficacy of theparticular vector, nucleic acid or immunogenic composition employed andthe condition of the patient, as well as the body weight or surface areaof the patient to be treated. The size of the dose also will bedetermined by the existence, nature, and extent of any adverseside-effects that accompany the administration of a particular vector,or transduced cell type in a particular patient.

In determining the effective amount of the nucleic acid, immunogeniccomposition or vector to be administered in the treatment or prophylaxisagainst Neisseria meningitidis, the physician evaluates circulatingplasma levels, vector and therepeutic moeity (e.g., anti-Neisseria mRNAribozyme) toxicities, progression of the disease, and the production ofanti-Neisseria meningitidis antibodies.

For administration, vectors, nucleic acids, immunogenic compositions andtransduced cells of the present invention can be administered at a ratedetermined by the LD-50 of the vector, immunogenic composition, ortransduced cell type, and the side-effects of the vector, nucleic acid,immunogenic composition, or cell type at various concentrations, asapplied to the mass and overall health of the patient. Administrationcan be accomplished via single or divided doses. For a typical 70 kgpatient, a dose equivalent to approximately 0.1 μg to 10 mg of vector ornucleic acid are administered. A dose of about 0.1 μg to 10 mg of mostimmunogenic compositions will suffice to elicit a protective immuneresponse against Neisseria meningitidis. In the case of immunogeniccompositions, booster inoculations of the immunogenic composition areoccasionally needed. Such booster inoculations are typicallyadministered from once every 5 years up to about four times per year.The need for a booster inoculation can be determined by measuring thelevel of anti-Neisseria meningitidis titer in the serum of theinoculated individual.

Transduced cells are optionally prepared for reinfusion according toestablished methods. See, Abrahamsen et al. (1991) J. Clin. Apheresis6:48-53; Carter et al. (1988) J. Clin. Apheresis 4:113-117; Aebersold etal. (1988), J. Immunol. Methods 112:1-7; Muul et al. (1987) J. Immunol.Methods 101:171-181 and Carter et al. (1987) Transfusion 27:362-365. Inone class of ex vivo procedures, between 1×10⁶ and 1×10⁹ transducedcells (e.g., stem cells, T cells or B cells transduced with vectorsencoding a nucleic acid of the invention) are infused intravenously,e.g., over 60-200 minutes. Vital signs and oxygen saturation by pulseoximetry are closely monitored. Blood samples are obtained 5 minutes and1 hour following infusion and saved for subsequent analysis.Leukopheresis, transduction and reinfusion may be repeated about every 2to 3 months for a total of 4 to 6 treatments in a one year period. Afterthe first treatment, infusions can be performed on a outpatient basis atthe discretion of the clinician.

If a patient undergoing infusion of a vector, immunogenic composition,or transduced cell develops fevers, chills, or muscle aches, he/shetypically receives the appropriate dose of aspirin, ibuprofen oracetaminophen. Patients who experience reactions to the infusion such asfever, muscle aches, and chills are premedicated 30 minutes prior to thefuture infusions with either aspirin, acetaminophen, or diphenhydramine.Meperidine is used for more severe chills and muscle aches that do notquickly respond to antipyretics and antihistamines. Cell infusion isslowed or discontinued depending upon the severity of the reaction.

The effect of the therapeutic vectors, immunogenic compositions, ortransduced cells of the invention on Neisseria meningitidis infectionand meningitis are measured by monitoring the level of Neisseriameningitidis in a patient, or by monitoring the anti-Neisseriameningitidis antibody count for the patient over time. Typically,measurements are taken before, during and after the therapeutic orprophylactic regimen.

EXAMPLES

The following examples are provided by way of illustration only and notby way of limitation. Those of skill will readily recognize a variety ofnoncritical parameters which can be changed or modified to yieldessentially similar results.

Example 1 ORF 1 (SEQ ID NO:2 ORF 2 (ORF 2a (SEQ ID NO:4), ORF 2b (SEQ IDNO:5)) and ORF 3 (SEQ ID NO:7) and Invasion Deficient Strains ofNeisseria meningitidis

Several hundred N. meningitidis serogroup B, strain NMB, Tn916transposon mutants were screened for an increased or decreased abilityto attach or invade human endometrial tissue culture (HEC-1-B) cells.Using this approach, we identified and characterized a mutant, VVV6,which showed a >10-fold decrease in its ability to invade HEC-1-B cellscompared to the parent NMB (strain) and to an additional wellcharacterized capsule deficient mutant, M7, (Stephens, D. S., et al.(1991), Infect. Immun., 59:4097-4102) (FIG. 2). The results obtainedfrom growth curves and the various controls used in theattachment-invasion assays revealed no significant difference in thegrowth rate between NMB and VVV6. The results of lipooligosaccharide(LOS) analysis from trains NMB, D1, M7, and VVV6 show that strain D1cannot sialylate LOS; this deficiency resulted in the loss of thesialilyted LOS band. There are no detectable differences in the LOSprofiles from NMB and VVV6. One dimensional SDS-PAGE analysis of outermembrane protein demonstrated that VVV6 had an identical profile to theparent strain. Electron microscopic analysis showed no difference in thequantity or morphology of the observable pili between NMB and VVV6.

Southern analysis on VVV6 genomic DNA digested with EcoRI, HindIII, andSau3A1 hybridized with a transposon specific probe showed band patternsconsistent with that of chromosomal DNA that contains only one copy ofthe transposon (Swartley, J. S., et al., (1993), Mol. Microbiol.,10:299-310). NMB has no Tn916 transposon inserted in the chromosome, andas expected there is no band observed in the Southern hybridization. Inaddition, DNA sequence analysis showed that the transposon insertion isof the Class 1 type (Hitchcock et al. (1983)); the entire transposon isinserted and stably maintained in the host genome (Swartley, J. S., etal., (1993), Mol. Microbiol., 10:299-310).

Linkage of the mutant phenotype with the location of the transposoninsertion was demonstrated by homologous recombination experiments.Transformation of the parent strain with genomic DNA from mutant VVV6yielded recombinants that showed the mutant phenotype when tested on thetissue culture monolayer (FIG. 3). Tetracycline resistantback-transformants were obtained at a frequency of 1.3×10⁵/mgDNA. Atotal of seven recombinants were tested, all of which showed a decreasedability to invade HEC-1-B cells. The polymerase chain reaction and DNAsequence analysis were used to determine the location of the transposoninsertion in each of the transformants. The results showed that thetransposon insertions occurred in the exact same position observed inVVV6.

Nucleotide sequence analysis on a 5 kb fragment showed that the Tn916insertion occurred between two open reading frames (Seq 3 (SEQ ID NO:6),encoding ORF 3 (SEQ ID NO:7), and Seq 2 (SEQ ID NO:3), encoding ORF-2,(ORF 2a (SEQ ID NO:4), ORF 2b (SEQ ID NO:5))). Seq 3 (SEQ ID NO:6) showsno significant homology to any gene in GenBank. Seq 2 (SEQ ID NO:3) is60%, identical to a gene in E. coli with no known function. Further DNAsequence analysis revealed a third open reading frame (Seq 1 (SEQ IDNO:1), encoding ORF 1 (SEQ ID NO:1)) downstream from Seq 2 (SEQ IDNO:3). The nucleotide sequence of Seq 1 (SEQ ID NO:1) is 61% identicalto the ftsZ gene from E. coli, a gene that has been shown to beessential for cell division and septum formation (See also, Example 2below).

The recent development of a transposon mutagenesis system (Buddingh, G.J., et al. (1987), Science, 86:20-21; Clark et al. (1987)) and the useof more appropriate virulence model systems provide the opportunity togain new insight into meningococcal disease. We have identified atransposon mutant, VVV6, that shows a decreased ability to invade HEC1-Btissue culture cells compared to the NMB parent strain and a wellcharacterized capsule deficient mutant, M7. Since both NMB and VVV6 showidentical replication rates in vitro, the lower numbers of viable countsobtained on the tissue culture assay are most likely due to a diminishedability of VVV6 to invade tissue culture cells.

The VVV6 strain produces identical lipopolysaccharide and SDS-PAGEprotein profiles and has similar quantity and type of pili compared toits parental strain. These data in conjunction with the fact thatcapsule analysis on VVV6 did not reveal any distinguishable differencescompared to the parent strain suggests that the mutation responsible forthe altered phenotype in mutant VVV6 is not likely due to capsuledeficiency or deficiency in any of the other major surface factors. Thedecreased ability of mutant VVV6 to invade HEC-1-B cells are linked tothe disruption of a gene(s) encoding for a factor(s) necessary forrecognition of a host cell receptor.

Bacteria and tissue culture cells.

N. meningitidis serogroup B strain NMB, and construction of theTn916-derived mutant library are described elsewhere (Buddingh, G. J.,et al., Science, 86:20-21). All Neisseria strains were maintained onCHOC-II agar (Carr-Scarborough, Atlanta, Ga.). The human endometrialcarcinoma cell line, HEC1-B, was maintained by the Biological ProductsBranch, CDC, Atlanta, Ga. Nearly confluent monolayers were maintained inminimal essential medium (MEM) with 10% fetal bovine serum (Gibco).

Attachment-Invasion Assay

Parent and Tn916 mutant strains were grown from frozen stocks on CHOC-IIagar plates to late log phase (18 hours) at 37° C. in 5% Co₂. The cellswere scraped and resuspended in MEM without serum to an OD₆₀₀ of 0.5(approximately 10⁸CFU/ml). Monolayers of Hec-1-B cells in 24 well disheswere infected using the resuspended cells. This produced a multiplicityof infection (MOI) of 10:1 (bacteria: host cell). Infection of tissueculture cells was allowed to proceed for 5 hours in 5% CO₂ at 37° C.After the infection was completed, each well was washed 5 times with MEMto remove most unattached bacterial cells.

To assay for attachment and invasion, 1 ml of MEM was added to eachwell. The monolayers were scraped, the suspension was diluted 10⁻³, and100 μl of this suspension was plated onto CHOCII agar and incubated withthe cells at 37° C. overnight. To assay for invasion only, 1 ml of MEMcontaining gentamicin (125 μg/ml) was added to each well after theinitial 5 hr assay and incubated at 37° C. in 5% CO₂ for 90 min.Monolayers were then washed twice with MEM. One-ml of MEM was added toeach well and the monolayers were scraped and diluted. Fifty-μl of thesuspension were plated on CHOCII agar plates. Plates were incubatedovernight at 37° C. in 5% CO₂.

Nasopharyngeal organ cultures.

Construction of the human nasopharyngeal organ culture model has beenpreviously described (Stephens, D. S., et al. (1991), Rev Infect Dis.,13:22-33). The model uses tissues obtained from children undergoingelective adenoidectomy, and allows quantitative and qualitativeassessment of the stages of attachment and invasion of N. meningitidisto the mucosal surface. This model system was used as a secondaryscreening method to evaluate the attachment and invasion properties ofthe mutant(s) identified using the HEC-1B monolayers. Nasopharyngealorgan cultures were incubated with meningococci. After 12 hours ofincubation the organ cultures were washed and the associated bacteriawere enumerated by homogenization of each organ culture and withsubsequent dilution and plating for colony counts.

Outer Membrane Protein Assay.

Meningococcal outer membrane proteins were isolated as described byClark et al, 1987. This method utilizes differential centrifugationfollowed by precipitation of outer membrane proteins in 2% sarcosyl. Thesamples were resolved on SDS-PAGE and the proteins visualized byCoomassie blue or silver stain.

Lipooligosacharide preparation.

LOS was prepared by lysis of bacteria in distilled water followed byproteinase K digestion as described by Hitchcock et al., 1983.

Electron microscopy of pili.

Negative staining grids of meningococci were prepared by fixation in 1%glutaraldehyde (cacodylate buffer) and staining with 1%phosphotungstate, and examination by transmission electron microscopy.

Southern Analysis.

Southern analysis was performed to demonstrate that Tn916 was insertedin the genomic DNA of mutant VVV6. A digoxigenin-labeled plasmidcontaining transposon Tn916 was used as a probe. Genomic DNA from VVV6and NMB was isolated and digested with the appropriate restrictionenzymes and transferred onto a nylon membrane (Kathariou, S., et al.Mol. Microbiol., 4:729-735). Hybridization was carried out as describedin the Genius System manual (Boehringer Mannheim Biochemicals). Briefly,blotted membranes were placed in hybridization tubes containing 20 ml ofprehybridization solution (5×SSC, 1% (w/v) blocking reagent, 0.1%N-laurysarcosine, 0.02% SDS) and incubated in a hybrization oven at 50°C. for at least 1 h. The prehybridization solution was replaced with 20ml of hybridization solution (prehybridization solution containing thedigoxygenin-labeled probe) and incubated over night at 52° C. Themembrane was washed 2× for 5 min with a 2×SSC buffer containing 0.1%SDS, then washed 3× for 5 min with a buffer consisting of 0.5× SSC and0.1% SDS. All washes were carried out at room temperature. Colorimetricdetection of DNA bands was performed as suggested by the manufacturer.

DNA amplification by PCR.

PCR was used to amplify chromosomal DNA fragments flanking Tn916. Thesequences at the ends of the transposon were previously reported(Clewell, D. B., et al. (1988), J. Bact., 170:3046-3052) and were usedin the design specific oligonucleotides that served as anchor primersfor PCR amplification of adjacent chromosomal DNA. Amplification andisolation of the unknown genomic DNA sequences was performed aspreviously described (Efrain M. Ribot, et al. (1996), Gene. Briefly,mutant genomic DNA was isolated and digested with Sau3A1 restrictionendonuclease. This restriction enzyme cuts both arms of the transposonnear the transposon-chromosome junction. After digestion was completed,the samples were phenol: chloroform-extracted, ethanol-precipitated andvacuum-desiccated using standard methodologies described by Sambrook etal. The DNA pellet was then resuspended in 20 ml of TE buffer, 2 ml ofSau3A linkers (250 mM/ml) and 2 μl of 10×T4 DNA ligase buffer and T4 DNAligase (10 units) were added. The ligation reaction was incubated atroom temperature for least 3 hours at. The samples were then phenol:chloroform extracted, ethanol precipitated and resuspended in 20 ml ofTE buffer.

The ligation mixture is then subjected to unidirectional PCRamplification (15 cycles:95° C.; 1 min, 52° C.; 1min, 72° C.; 1½ min in25 ml volumes) of the target DNA using 5′ biotin-labeled anchor primersspecific for the known sequences of the right arm and left arm of thetransposon. The resulting single-stranded PCR product contained theadjacent unknown chromosomal DNA flanked by the remaining portion of thetransposon and the sequences corresponding to the ligated linker. Thebiotin-labeled single-stranded PCR (ssPCR) product containing theflanking chromosomal DNA was captured using streptaiving-coated beads asdescribed by the manufacturer (Dynal AS, Oslo, Norway).

The particle-isolated ssPCR products were subjected to 25 cycles of PCRamplification (94° C.:1 min; 50° C.:30 sec; 72° C.:1½ min in 25 μlvolumes). Transposon and linker specific primers were used for thispurpose. The resulting PCR fragments were cloned or sequenced directlyas described by Ribot et al., Manuscript submitted for publication. Allthe oligonucleotide primers used in this study were synthesized by theCDC Biotechnology Core Facility.

DNA sequencing.

Automated DNA sequence analysis was performed using both the Sangerdideoxy method (Amplitaq for sequencing, Perking-Elmer, Foster City,Calif.) and the dye terminator reaction method as described in the ABIinstruction manual.

Example 2 The Neisseria Meningitidis ftsZ Homologue

The nucleotide sequence of a 1.2 kb DNA fragment of Neisseriameningitidis DNA that contains an open reading frame (Seq 1 (SEQ IDNO:1), encoding ORF 1 (SEQ ID NO:2)) that is highly homologous to thecorresponding ORF from the Escherichia coli ftsZ gene is described inthis example. The E. coli ftsZ gene codes for a GTP-binding proteinessential for septum formation and cell division. The 1.2 kb N.meningitidis ORF 1 is 61% identical, at the nucleotide sequence level,to the ftsZ gene of E. coli and 50% identical at the amino acid level.The predicted polypeptide contains a glycine-rich stretch of seven aminoacids that is identical to the highly conserved GTP-binding domain foundin all the ftsZ genes identified thus far. Based on these data, Seq 1(SEQ ID NO:1) codes for the N. meningitidis cell division protein FtsZ.

DNA amplification by PCR.

Neisseria meningitidis mutant and wild-type strains were grown on CHOCIIagar (Carr-Scarborough, Atlanta, Ga.) plates at 37° C. in 5% CO₂ overnight. Genomic DNA was isolated using the Isoquick nucleic acidextraction kit (ORCA Research Inc., Bothell, Wash.) under the conditionsdescribed by the manufacturer. The procedure used for the amplificationof chromosomal DNA fragments was based on a method developed for therapid amplification of transposon ends (RATE). A modified version ofRATE was used to chromosome walk up- land downstream from the transposoninsertion site in mutant VVV6. Briefly, genomic DNA was isolated fromthe bacterial strain and 5 μg digested with the desired restrictionendonuclease. The restriction enzyme HindIII was used. After digestionwas completed, the sample was phenol: chloroform treated and vacuumusing standard methods (Sambrook et al., 1989). The pellet containingthe total genomic digest is resuspended in 15 μl of double distilledsterile H₂O and 2 ml of the appropriate linkers (250 mM/ml), 10 units ofDNA ligase, and 2.5 μl of 10×T4 DNA ligase buffer added and the samplevolume adjusted to 25 ml with double-distilled sterile water. Theligation reaction was then allowed to proceed for at least three hoursat room temperature. Construction of the HIEC linker was done by addingequimolar amounts of each oligonucleotide, HEIC1(ATCTTGAGGTCGACGGGATATCG) (SEQ ID NO:10) and HEIC2(AATTCGATATCCCGTCGACCTCA) (SEQ ID NO:11), incubating at 90° C. for 5 minand allowing the samples to cool slowly to room temperature. Excesslinkers are removed by passing the samples through Microcon 100 filtersas described by the manufacturer (Amicon Inc., Beverly, Mass.).

Unidirectional PCR amplification (15 cycles: 95° C.; 1 min, 52° C.;1min, 72° C.;1½ min in 25 ml volumes) of the target sequence wasperformed using a 5′ biotin=labeled primer/reaction (B800F1CACATAAGGCGTGGTGGAAG (SEQ ID NO:12) )) specific for the known genomicsequence obtained from previous sequencing reactions. Thisunidirectional amplification reaction yields single-stranded DNAmolecules containing the chromosomal target sequence, the adjacentunknown chromosomal DNA, and the linker. Streptavidin coated beads(Dynal AS, Oslo, Norway) were used to capture the PCR-amplifiedbiotin-labeled single-stranded products following the manufacturersrecommendations. Aliqots of the purified single-stranded PCR productswere then subjected to 30 cycles of PCR amplification (94° C.:1 min; 42°C.:30 sec; 72° C.:1½ min in 25 ml volumes), using a nested primerspecific for the for the known sequence (800F8 CTCCCAAACCGGACAAACCG (SEQID NO:13)) and a primer corresponding to the ligated linker (HIEC2). A 5ml aliquot of each of the resulting double-stranded PCR products wasloaded onto a 0.8% agarose gel to determine product size and purity(data not shown). Selected products were then subjected to automated DNAsequence analysis using primers specific to both the known genomic(800F9 GTCAAGTACGGACTGATTGTCG (SEQ ID NO:14)) sequence and the HEIC2linker primer.

DNA sequencing.

Automated DNA sequence analysis of PCR amplified fragments was performedusing the dye terminator reaction method as described in the ABI-373instruction manual (Perking-Elmer, Foster City, Calif.). Computerassisted analysis was performed using the Wisconsin Sequence AnalysisPackage (GCG) (Madison, Wis.) and DNASIS, (National Bioscience, Inc,Plymouth, Minn.).

The Tn916 transposon mutant of N. meningitidis, serogroup B, strain NMB,demonstrated a significant decrease in its ability to invade humanepithelial tissue culture cells compared to control strains. Sequencinganalysis on VVV6 genomic DNA indicated that the transposon insertionoccurred between two possible open reading frames (Seq 3 (SEQ ID NO:6)and Seq 2 (SEQ ID NO:3)) (FIG. 1). Further DNA sequence analysis on theregion downstream from Seq 2 (SEQ ID NO:3) revealed a another ORF (Seq 1(SEQ ID NO:1)). Nucleotide sequence comparison of this ORF (Seq 1 (SEQID NO:1)) using the FASTA algorithm of the GCG Wisconsin package showsthat the nucleotide sequence of Seq 1 (SEQ ID NO:1) is over 61%identical to the E. coli essential cell division gene ftsZ. All ftsZgenes identified to date show a high degree of homology. We have alsoidentified both a possible ribosome binding site and start codon forthis ORF (Seq 1 (SEQ ID NO:1)) and there are two possible stop codons atnucleotide positions 1100 and 1148. Primer extension and S1 nucleaseprotection studies are used to determine the precise location ofpromoter regions and termination sequences of Seq 1 (SEQ ID NO:1), Seq 2(SEQ ID NO:3) and Seq 3 (SEQ ID NO:6).

The amino acid sequence of the ORF 1 polypeptide (SEQ ID NO:1) is 50%identical to the FtsZ protein from E. coli and B. subtilis. Furthermore,the amino acid sequence of the N. meningitidis FtsZ protein contains thehighly conserved GTP-binding domain present in all the FtsZ proteinsidentified thus far (de Boer, et al. (1992) Nature 359:254-56;Mukherjee, et al. (1993) Proc. Natl. Acad. Sci. USA. 90:1053-57; Beall,et al. (1988) J. Bacteriol. 170:4855-4864).

A highly conserved glycine-rich stretch of amino acids (GGGTGTG (SEQ IDNO:15)) has been found in all the FtsZ proteins identified so far(Corton, et al. (1987) J. Bacteriol. 169:1-7; de Boer, et al. (1992)Nature 359:254-56). As can be observed from amino acid residues atapproximately 109 to 115 of ORF 1 (SEQ ID NO:1), the amino acid sequenceof the polypeptide encoded by ORF 1 (SEQ ID NO:1) also contains thishighly conserved domain. This provides additional evidence that the geneproduct encoded by the Neisseria ORF is the homolog of the FtsZ proteinfrom E. coli. In vitro assays indicate that this glycine-rich sequencecontains a domain with GTP/GDP-binding activity (Corton, et al. (1987)J. Bacteriol. 169:1-7; de Boer, et al. (1992) Nature 359:254-56;Mukherjee, et al. (1993) Proc. Natl. Acad. Sci. USA. 90:1053-57).Escherichia coli cells have been characterized that carry mutationswithin this amino acid stretch that result in a cell division deficientphenotype. The inability of such mutants to divide has been linked toreduced GTPase activity (Cook, et al. (1994) Mol. Microbiol. 14:485-495;Ricard, et al. (1973) J. Bacteriol. 116:314-322). It has beendemonstrated that the E. coli functional unit of FtsZ consists ofmultiple copies of FtsZ assembled together in a multimeric complex. Itappears that the GTPase activity is required for the assembly of such acomplex. If a mutated FtsZ has a decreased ability to bind GTP, complexformation will not occur as it would under normal conditions, thusdiminishing the cell's ability to divide. This stretch of amino acids isnot only conserved among the eubacteria (Lutkenhaus, et al. (1980) J.Bacteriol. 142:615-620; Miyakawa, et al. (1972) J. Bacteriol112:959-958), but is also remarkably similar to the a-, b-, andg-tubulins from eukaryotic cells (Gill, et al. (1986) Mol. Gen. Genet.205:134-145). FtsZ may be the predecessor of the more evolutionarilyrecent tubulin (Bermudez, et al. (1994) Microbiol. Rev. 58:387-400).This hypothesis is supported by the recent discovery of an ftsZ homologgene from the archaebacterium Halobacterium salinarum. Amino acidsequence aligment of the H. salinarum FtsZ showed remarkable similarityto the FtsZ proteins from eubacteria and tubulins from eucaryotic cells.

In E. coli, ftsZ is preceded by the ftsA gene and followed by the envAgene. The nucleotide sequence of a 225 bp long segment of DNA upstreamof ORF 1 (SEQ ID NO:7) from N. meningitidis, NMB, was obtained, butfailed to reveal any significant homology to the ftsA gene from E. coli.The DNA sequence downstream of the Neisseria ftsZ also revealed nohomology to the E. coli envA gene. This is not surprising since the DNAregions flanking the ftsZ gene from organisms such as Bacillus subtilis(Beall, et al. (1988) J. Bacteriol. 170:4855-4864), Streptomycescoleicolor (McCormick, et al. (1994) Mol. Microbiol. 14:243-254), and H.salinarum (Margolin, et al. (1996) J. Bacteriol. 178:1320-1327) do notshow the same genetic map observed in E. coli.

While a hypothetical ribosome binding site (RBS) and start codon (ATG)were found, no obvious consensus promoter sequence was identified inassociation with the ftsZ-homolog gene. This ORF may be controlled by apromoter located elsewhere in the DNA region upstream; in E. coli, thepromoter controlling expression of ftsZ is found upstream within theftsA gene. Primer extension analysis ultimately defines the start siteof transcription. In addition, there is no obvious termination sequenceat the end of the ORF of the ftsZ-homolog, suggesting that the gene isexpressed as part of a polycistronic message in Neisseria meningitidis.Interestingly, computer analysis revealed a strong termination loop atthe end of Seq 2 (SEQ ID NO:3); this may indicate the end oftranscription of the polygenic mRNA. Again, this genetic arrangementbears a strong resemblance to the ftsZ gene region from E. coli., whichconsists of an operon-like structure containing the ftsQ, ftsA, ftsZ,and envA genes.

Discussion of the Accompanying Sequence Listing

SEQ ID NO:8 provides the sequence of Seq 4. This sequence encompassesSeq 1, Seq 2, and Seq 3, which are additionally provided at SEQ ID NO:1,SEQ ID NO:3, and SEQ ID NO:6, respectively. The information for thenucleic acid sequences are presented as DNA sequence information. One ofskill will readily understand that portions of the sequences alsodescribe RNAs encoded by the sequence (e.g, by substitution of Tresidues with corresponding U residues), and a variety of conservativelymodified variations, including silent substitutions of the sequences.While only a single strand of sequence information is shown, one ofskill will immediately appreciate that the complete correspondingcomplementary sequence is fully described by comparison to the givensequences.

SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:7 provide the aminoacid sequences of ORF 1, ORF 2a, ORF 2b, and ORF 3, respectively. Avariety of conservatively modified variations of the amino acidsequences provided will be apparent to one of skill, and are describedherein. One of skill will also recognize that a variety of nucleic acidsequences encode each of the polypeptides due to the codon degeneracypresent in the genetic code. Each of the nucleic acids which encodes thegiven polypeptide is described by comparison to the amino acid sequenceand translation via the genetic code.

The above examples are provided to illustrate the invention but not tolimit its scope. Other variants of the invention will be readilyapparent to one of ordinary skill in the art and are encompassed by theappended claims. All publications, patents, and patent applicationscited herein are hereby incorporated by reference for all purposes.

SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 15 <210> SEQ ID NO 1 <211>LENGTH: 1185 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence;Note = synthetic construct <221> NAME/KEY: CDS <222> LOCATION:(17)...(1102) <221> NAME/KEY: misc_feature <222> LOCATION: (1)...(1185)<223> OTHER INFORMATION: Note:/Seq 1 = position 223 through position1407 of Seq 4 <221> NAME/KEY: misc_feature <222> LOCATION: (17)...(1102)<223> OTHER INFORMATION: product = ORF 1 Note:/ = ORF 1 CDS = position238 through position 1324 of Seq 4 <400> SEQUENCE: 1 gagcaggagt ttttgaatg gaa ttt gtt tac gac gtg gca gaa tcg gca gtc 52 Met Glu Phe Val TyrAsp Val Ala Glu Ser Ala Val 1 5 10 agc cct gcg gtg att aaa gta atc ggcttg ggc ggc ggc ggt tgc aat 100 Ser Pro Ala Val Ile Lys Val Ile Gly LeuGly Gly Gly Gly Cys Asn 15 20 25 gca tcc aat aac atg gtt gcc aac aat gtgcgc ggt gtg gag ttt atc 148 Ala Ser Asn Asn Met Val Ala Asn Asn Val ArgGly Val Glu Phe Ile 30 35 40 agt gcc aat acg gat gcg cag tct ctg gca aaaaac cat gcg gcg aag 196 Ser Ala Asn Thr Asp Ala Gln Ser Leu Ala Lys AsnHis Ala Ala Lys 45 50 55 60 aga atc cag ttg ggt acg aat ctg aca cgc ggtttg ggc gcg ggc gcg 244 Arg Ile Gln Leu Gly Thr Asn Leu Thr Arg Gly LeuGly Ala Gly Ala 65 70 75 aat ccc gat atc ggc cgt gcg gca gcc cag gaa gaccgg gaa gcc att 292 Asn Pro Asp Ile Gly Arg Ala Ala Ala Gln Glu Asp ArgGlu Ala Ile 80 85 90 gaa gaa gcc att cgc ggt gcg aat atg ctg ttt atc acgacc ggt atg 340 Glu Glu Ala Ile Arg Gly Ala Asn Met Leu Phe Ile Thr ThrGly Met 95 100 105 ggc ggc ggt acc ggt acc ggt tcc gcg ccg gtt gtt gctgag att gcc 388 Gly Gly Gly Thr Gly Thr Gly Ser Ala Pro Val Val Ala GluIle Ala 110 115 120 aag tct ttg ggc att ctg acc gtt gcc gtg gtt acc cgaccg ttc gca 436 Lys Ser Leu Gly Ile Leu Thr Val Ala Val Val Thr Arg ProPhe Ala 125 130 135 140 tat gaa ggt aag cgc gtc cat gtc gca cag gca gggttg gaa cag ttg 484 Tyr Glu Gly Lys Arg Val His Val Ala Gln Ala Gly LeuGlu Gln Leu 145 150 155 aaa gaa cac gtc gat tcg ctg att atc atc ccg aacgac aaa ctg atg 532 Lys Glu His Val Asp Ser Leu Ile Ile Ile Pro Asn AspLys Leu Met 160 165 170 act gca ttg ggt gaa gac gta acg atg cgc gaa gccttc cgt gcc gcc 580 Thr Ala Leu Gly Glu Asp Val Thr Met Arg Glu Ala PheArg Ala Ala 175 180 185 gac aat gta ttg cgc gat gcg gtc gca ggc att tccgaa gtg gta act 628 Asp Asn Val Leu Arg Asp Ala Val Ala Gly Ile Ser GluVal Val Thr 190 195 200 tgc ccg agc gaa atc atc aac ctc gac ttt gcc gacgtg aaa acc gtg 676 Cys Pro Ser Glu Ile Ile Asn Leu Asp Phe Ala Asp ValLys Thr Val 205 210 215 220 atg agc aac cgc ggt atc gct atg atg ggt tcgggt tat gcc caa ggt 724 Met Ser Asn Arg Gly Ile Ala Met Met Gly Ser GlyTyr Ala Gln Gly 225 230 235 atc gac cgt gcg cgt atg gcg acc gac cag gccatt tcc agt ccg ctg 772 Ile Asp Arg Ala Arg Met Ala Thr Asp Gln Ala IleSer Ser Pro Leu 240 245 250 ctg gac gat gta acc ttg gac gga gcg cgc ggtgtg ctg gtc aat att 820 Leu Asp Asp Val Thr Leu Asp Gly Ala Arg Gly ValLeu Val Asn Ile 255 260 265 acg act gct ccg ggt tgc ttg aaa atg tcc gagttg tcc gaa gtc atg 868 Thr Thr Ala Pro Gly Cys Leu Lys Met Ser Glu LeuSer Glu Val Met 270 275 280 aaa atc gtc aac caa agc gcg cat ccc gat ttggaa tgc aaa ttc ggt 916 Lys Ile Val Asn Gln Ser Ala His Pro Asp Leu GluCys Lys Phe Gly 285 290 295 300 gct gct gaa gac gag acc atg agc gaa gatgcc atc cgg att acc att 964 Ala Ala Glu Asp Glu Thr Met Ser Glu Asp AlaIle Arg Ile Thr Ile 305 310 315 atc gct acc ggt ctg aaa gaa aaa ggc gcggtc gat ttt gtt ccg gca 1012 Ile Ala Thr Gly Leu Lys Glu Lys Gly Ala ValAsp Phe Val Pro Ala 320 325 330 agg gag gta gaa gcg gtt gcc ccg tcc aaacag gag caa agc cac aat 1060 Arg Glu Val Glu Ala Val Ala Pro Ser Lys GlnGlu Gln Ser His Asn 335 340 345 gtc gaa ggt aga tcc gca cca atc gcg gtatcc gca cga tga 1102 Val Glu Gly Arg Ser Ala Pro Ile Ala Val Ser AlaArg * 350 355 360 accttaccgc tgcggatttc gacaatcagt ccgtacttga cgacttgaaatccctgcgat 1162 tttgcgtcgt caacacaatt cag 1185 <210> SEQ ID NO 2 <211>LENGTH: 361 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence;Note = synthetic construct <400> SEQUENCE: 2 Met Glu Phe Val Tyr Asp ValAla Glu Ser Ala Val Ser Pro Ala Val 1 5 10 15 Ile Lys Val Ile Gly LeuGly Gly Gly Gly Cys Asn Ala Ser Asn Asn 20 25 30 Met Val Ala Asn Asn ValArg Gly Val Glu Phe Ile Ser Ala Asn Thr 35 40 45 Asp Ala Gln Ser Leu AlaLys Asn His Ala Ala Lys Arg Ile Gln Leu 50 55 60 Gly Thr Asn Leu Thr ArgGly Leu Gly Ala Gly Ala Asn Pro Asp Ile 65 70 75 80 Gly Arg Ala Ala AlaGln Glu Asp Arg Glu Ala Ile Glu Glu Ala Ile 85 90 95 Arg Gly Ala Asn MetLeu Phe Ile Thr Thr Gly Met Gly Gly Gly Thr 100 105 110 Gly Thr Gly SerAla Pro Val Val Ala Glu Ile Ala Lys Ser Leu Gly 115 120 125 Ile Leu ThrVal Ala Val Val Thr Arg Pro Phe Ala Tyr Glu Gly Lys 130 135 140 Arg ValHis Val Ala Gln Ala Gly Leu Glu Gln Leu Lys Glu His Val 145 150 155 160Asp Ser Leu Ile Ile Ile Pro Asn Asp Lys Leu Met Thr Ala Leu Gly 165 170175 Glu Asp Val Thr Met Arg Glu Ala Phe Arg Ala Ala Asp Asn Val Leu 180185 190 Arg Asp Ala Val Ala Gly Ile Ser Glu Val Val Thr Cys Pro Ser Glu195 200 205 Ile Ile Asn Leu Asp Phe Ala Asp Val Lys Thr Val Met Ser AsnArg 210 215 220 Gly Ile Ala Met Met Gly Ser Gly Tyr Ala Gln Gly Ile AspArg Ala 225 230 235 240 Arg Met Ala Thr Asp Gln Ala Ile Ser Ser Pro LeuLeu Asp Asp Val 245 250 255 Thr Leu Asp Gly Ala Arg Gly Val Leu Val AsnIle Thr Thr Ala Pro 260 265 270 Gly Cys Leu Lys Met Ser Glu Leu Ser GluVal Met Lys Ile Val Asn 275 280 285 Gln Ser Ala His Pro Asp Leu Glu CysLys Phe Gly Ala Ala Glu Asp 290 295 300 Glu Thr Met Ser Glu Asp Ala IleArg Ile Thr Ile Ile Ala Thr Gly 305 310 315 320 Leu Lys Glu Lys Gly AlaVal Asp Phe Val Pro Ala Arg Glu Val Glu 325 330 335 Ala Val Ala Pro SerLys Gln Glu Gln Ser His Asn Val Glu Gly Arg 340 345 350 Ser Ala Pro IleAla Val Ser Ala Arg 355 360 <210> SEQ ID NO 3 <211> LENGTH: 960 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence; Note = syntheticconstruct <221> NAME/KEY: misc_feature <222> LOCATION: (1)...(960) <223>OTHER INFORMATION: Note:/ Seq 2 = positions 1921 through 2880 of Seq 4<221> NAME/KEY: misc_feature <222> LOCATION: (39)...(941) <223> OTHERINFORMATION: /product = ORF 2a Note:/ ORF 2 protein variant usingalternate start site at position 39 of Seq 2 (position 1959 throughposition 2861 of Seq 4) <221> NAME/KEY: misc_feature <222> LOCATION:(51)...(941) <223> OTHER INFORMATION: / product = ORF 2b Note:/ ORF 2protein variant using alternate start site at position 51 of Seq 2(position 1971 through position 2861 of Seq 4) <400> SEQUENCE: 3ttttttaaag tcagggaaat gctgtcaacg cactgcct atg ggt ttg aaa atg tcg 56 MetGly Leu Lys Met Ser 1 5 att gct gcc ggt atc ggt ttg ttt ttg gca ctg atttcc ctg aaa ggc 104 Ile Ala Ala Gly Ile Gly Leu Phe Leu Ala Leu Ile SerLeu Lys Gly 10 15 20 gca ggc cat tat cgt tgc caa tcc ggc aac ctt ggt cggttt ggg cga 152 Ala Gly His Tyr Arg Cys Gln Ser Gly Asn Leu Gly Arg PheGly Arg 25 30 35 tat tca tca gcc gtc cgc gtt gtt ggc act gtt cgg ttt tgctat ggt 200 Tyr Ser Ser Ala Val Arg Val Val Gly Thr Val Arg Phe Cys TyrGly 40 45 50 ggt cgt att ggg aca ttt ccg cgt tca agg cgc aac atc atc accatc 248 Gly Arg Ile Gly Thr Phe Pro Arg Ser Arg Arg Asn Ile Ile Thr Ile55 60 65 70 ttg acc att acc gtc att gcc agc ctg atg ggt ttg aat gaa tttcac 296 Leu Thr Ile Thr Val Ile Ala Ser Leu Met Gly Leu Asn Glu Phe His75 80 85 ggc atc atc ggc gaa gta ccg agc att gcg ccg act ttt atg cag atg344 Gly Ile Ile Gly Glu Val Pro Ser Ile Ala Pro Thr Phe Met Gln Met 9095 100 gat ttt gaa ggc ctg ttt acc gtc agc tgg tca gtg att ttc gtc ttc392 Asp Phe Glu Gly Leu Phe Thr Val Ser Trp Ser Val Ile Phe Val Phe 105110 115 ttc ttg gtc gat cta ttt gac agt acc gga acg ctg gtc ggc ata tcc440 Phe Leu Val Asp Leu Phe Asp Ser Thr Gly Thr Leu Val Gly Ile Ser 120125 130 cac cgt gcc ggg ctg ctg gtg gac ggt aag ctg ccc cgc ctg aaa cgc488 His Arg Ala Gly Leu Leu Val Asp Gly Lys Leu Pro Arg Leu Lys Arg 135140 145 150 gca ctg ctt gca gac tct acc gcc att atg gca ggt gcg gct ttgggt 536 Ala Leu Leu Ala Asp Ser Thr Ala Ile Met Ala Gly Ala Ala Leu Gly155 160 165 act tct tcc acc acg cct tat gtg gaa agc gcg gcg ggc gta tcggca 584 Thr Ser Ser Thr Thr Pro Tyr Val Glu Ser Ala Ala Gly Val Ser Ala170 175 180 ggc gga cgg acc ggc ctg acg gcg gtt acc gtc ggc gta ttg atgctc 632 Gly Gly Arg Thr Gly Leu Thr Ala Val Thr Val Gly Val Leu Met Leu185 190 195 gcc tgc ctg atg ttt tca cct ttg gcg aaa agt gtt ccc gct tttggc 680 Ala Cys Leu Met Phe Ser Pro Leu Ala Lys Ser Val Pro Ala Phe Gly200 205 210 acc gcg ccc gcc ctg ctt tat gtc ggc acg cag atg ctc cgc agtgcg 728 Thr Ala Pro Ala Leu Leu Tyr Val Gly Thr Gln Met Leu Arg Ser Ala215 220 225 230 agg gat att gat tgg gac gat atg acg gaa gcc gca ccc gcattc ctg 776 Arg Asp Ile Asp Trp Asp Asp Met Thr Glu Ala Ala Pro Ala PheLeu 235 240 245 acc att gtc ttc atg ccg ttt acc tat tcg att gca gac ggcatc gcc 824 Thr Ile Val Phe Met Pro Phe Thr Tyr Ser Ile Ala Asp Gly IleAla 250 255 260 ttc ggc ttc atc agc tat gcc gtg gtt aaa ctt tta tgc cgccgc acc 872 Phe Gly Phe Ile Ser Tyr Ala Val Val Lys Leu Leu Cys Arg ArgThr 265 270 275 aaa gac gtt ccg cct atg gaa tgg gtt gtt gcc gta ttg tgggca ctg 920 Lys Asp Val Pro Pro Met Glu Trp Val Val Ala Val Leu Trp AlaLeu 280 285 290 aaa ttc tgg tat ttg ggc tga ttgattcgat attaaaaat 960 LysPhe Trp Tyr Leu Gly * 295 300 <210> SEQ ID NO 4 <211> LENGTH: 300 <212>TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence; Note = syntheticconstruct <400> SEQUENCE: 4 Met Gly Leu Lys Met Ser Ile Ala Ala Gly IleGly Leu Phe Leu Ala 1 5 10 15 Leu Ile Ser Leu Lys Gly Ala Gly His TyrArg Cys Gln Ser Gly Asn 20 25 30 Leu Gly Arg Phe Gly Arg Tyr Ser Ser AlaVal Arg Val Val Gly Thr 35 40 45 Val Arg Phe Cys Tyr Gly Gly Arg Ile GlyThr Phe Pro Arg Ser Arg 50 55 60 Arg Asn Ile Ile Thr Ile Leu Thr Ile ThrVal Ile Ala Ser Leu Met 65 70 75 80 Gly Leu Asn Glu Phe His Gly Ile IleGly Glu Val Pro Ser Ile Ala 85 90 95 Pro Thr Phe Met Gln Met Asp Phe GluGly Leu Phe Thr Val Ser Trp 100 105 110 Ser Val Ile Phe Val Phe Phe LeuVal Asp Leu Phe Asp Ser Thr Gly 115 120 125 Thr Leu Val Gly Ile Ser HisArg Ala Gly Leu Leu Val Asp Gly Lys 130 135 140 Leu Pro Arg Leu Lys ArgAla Leu Leu Ala Asp Ser Thr Ala Ile Met 145 150 155 160 Ala Gly Ala AlaLeu Gly Thr Ser Ser Thr Thr Pro Tyr Val Glu Ser 165 170 175 Ala Ala GlyVal Ser Ala Gly Gly Arg Thr Gly Leu Thr Ala Val Thr 180 185 190 Val GlyVal Leu Met Leu Ala Cys Leu Met Phe Ser Pro Leu Ala Lys 195 200 205 SerVal Pro Ala Phe Gly Thr Ala Pro Ala Leu Leu Tyr Val Gly Thr 210 215 220Gln Met Leu Arg Ser Ala Arg Asp Ile Asp Trp Asp Asp Met Thr Glu 225 230235 240 Ala Ala Pro Ala Phe Leu Thr Ile Val Phe Met Pro Phe Thr Tyr Ser245 250 255 Ile Ala Asp Gly Ile Ala Phe Gly Phe Ile Ser Tyr Ala Val ValLys 260 265 270 Leu Leu Cys Arg Arg Thr Lys Asp Val Pro Pro Met Glu TrpVal Val 275 280 285 Ala Val Leu Trp Ala Leu Lys Phe Trp Tyr Leu Gly 290295 300 <210> SEQ ID NO 5 <211> LENGTH: 296 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence; Note = synthetic construct <400>SEQUENCE: 5 Met Ser Ile Ala Ala Gly Ile Gly Leu Phe Leu Ala Leu Ile SerLeu 1 5 10 15 Lys Gly Ala Gly His Tyr Arg Cys Gln Ser Gly Asn Leu GlyArg Phe 20 25 30 Gly Arg Tyr Ser Ser Ala Val Arg Val Val Gly Thr Val ArgPhe Cys 35 40 45 Tyr Gly Gly Arg Ile Gly Thr Phe Pro Arg Ser Arg Arg AsnIle Ile 50 55 60 Thr Ile Leu Thr Ile Thr Val Ile Ala Ser Leu Met Gly LeuAsn Glu 65 70 75 80 Phe His Gly Ile Ile Gly Glu Val Pro Ser Ile Ala ProThr Phe Met 85 90 95 Gln Met Asp Phe Glu Gly Leu Phe Thr Val Ser Trp SerVal Ile Phe 100 105 110 Val Phe Phe Leu Val Asp Leu Phe Asp Ser Thr GlyThr Leu Val Gly 115 120 125 Ile Ser His Arg Ala Gly Leu Leu Val Asp GlyLys Leu Pro Arg Leu 130 135 140 Lys Arg Ala Leu Leu Ala Asp Ser Thr AlaIle Met Ala Gly Ala Ala 145 150 155 160 Leu Gly Thr Ser Ser Thr Thr ProTyr Val Glu Ser Ala Ala Gly Val 165 170 175 Ser Ala Gly Gly Arg Thr GlyLeu Thr Ala Val Thr Val Gly Val Leu 180 185 190 Met Leu Ala Cys Leu MetPhe Ser Pro Leu Ala Lys Ser Val Pro Ala 195 200 205 Phe Gly Thr Ala ProAla Leu Leu Tyr Val Gly Thr Gln Met Leu Arg 210 215 220 Ser Ala Arg AspIle Asp Trp Asp Asp Met Thr Glu Ala Ala Pro Ala 225 230 235 240 Phe LeuThr Ile Val Phe Met Pro Phe Thr Tyr Ser Ile Ala Asp Gly 245 250 255 IleAla Phe Gly Phe Ile Ser Tyr Ala Val Val Lys Leu Leu Cys Arg 260 265 270Arg Thr Lys Asp Val Pro Pro Met Glu Trp Val Val Ala Val Leu Trp 275 280285 Ala Leu Lys Phe Trp Tyr Leu Gly 290 295 <210> SEQ ID NO 6 <211>LENGTH: 457 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (17)...(457) <221>NAME/KEY: misc_feature <222> LOCATION: (1)...(457) <223> OTHERINFORMATION: Note:/ n = a,t,c, or g <221> NAME/KEY: misc_feature <222>LOCATION: (1)...(457) <223> OTHER INFORMATION: Note:/ Seq 3 = position3381 through position 3837 of Seq 4 <221> NAME/KEY: misc_feature <222>LOCATION: (17)...(457) <223> OTHER INFORMATION: / product = ORF 3 Note:/ORF 3 CDS = position 3397 through position 3837 of Seq 4 <400> SEQUENCE:6 taatgattgg attggg atg ccc gac gcg tcg gat ggc tgt gtt ttg ccg tcc 52Met Pro Asp Ala Ser Asp Gly Cys Val Leu Pro Ser 1 5 10 gaa tgt gat ggaagc ctg tcc ata ctg aaa aaa agt cta tan agg aga 100 Glu Cys Asp Gly SerLeu Ser Ile Leu Lys Lys Ser Leu Xaa Arg Arg 15 20 25 aat atg atg agt caacac tct gcc gga gca cgt ttc cgc caa gcc gtg 148 Asn Met Met Ser Gln HisSer Ala Gly Ala Arg Phe Arg Gln Ala Val 30 35 40 aaa gaa tcg aat ccg cttgcc gtc gcc ggt tgc gtc aat gct tat ttt 196 Lys Glu Ser Asn Pro Leu AlaVal Ala Gly Cys Val Asn Ala Tyr Phe 45 50 55 60 gca cga ttg gcc acc caaagc ggt ttc aaa gcc atc tat ctg tct ggc 244 Ala Arg Leu Ala Thr Gln SerGly Phe Lys Ala Ile Tyr Leu Ser Gly 65 70 75 ggc ggc gtg gca gcc tgt tcttgc ggt atc cct gat ttg ggc att acc 292 Gly Gly Val Ala Ala Cys Ser CysGly Ile Pro Asp Leu Gly Ile Thr 80 85 90 aca atg gaa gat gtg ctg atc gacgca cga cgc att acg gac aac gtg 340 Thr Met Glu Asp Val Leu Ile Asp AlaArg Arg Ile Thr Asp Asn Val 95 100 105 gat ncg cct ctg ctg gtg gac atcgat gtg ggt tgg ggc ggt gca ttc 388 Asp Xaa Pro Leu Leu Val Asp Ile AspVal Gly Trp Gly Gly Ala Phe 110 115 120 aat att gcc cgt acc att cgc aacttt gaa cgc gcc ggt gtt gca gcg 436 Asn Ile Ala Arg Thr Ile Arg Asn PheGlu Arg Ala Gly Val Ala Ala 125 130 135 140 gtt cac atc gaa gat cag gta457 Val His Ile Glu Asp Gln Val 145 <210> SEQ ID NO 7 <211> LENGTH: 147<212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence; Note = syntheticconstruct <221> NAME/KEY: VARIANT <222> LOCATION: (1)...(147) <223>OTHER INFORMATION: Note: Xaa = any amino acid <400> SEQUENCE: 7 Met ProAsp Ala Ser Asp Gly Cys Val Leu Pro Ser Glu Cys Asp Gly 1 5 10 15 SerLeu Ser Ile Leu Lys Lys Ser Leu Xaa Arg Arg Asn Met Met Ser 20 25 30 GlnHis Ser Ala Gly Ala Arg Phe Arg Gln Ala Val Lys Glu Ser Asn 35 40 45 ProLeu Ala Val Ala Gly Cys Val Asn Ala Tyr Phe Ala Arg Leu Ala 50 55 60 ThrGln Ser Gly Phe Lys Ala Ile Tyr Leu Ser Gly Gly Gly Val Ala 65 70 75 80Ala Cys Ser Cys Gly Ile Pro Asp Leu Gly Ile Thr Thr Met Glu Asp 85 90 95Val Leu Ile Asp Ala Arg Arg Ile Thr Asp Asn Val Asp Xaa Pro Leu 100 105110 Leu Val Asp Ile Asp Val Gly Trp Gly Gly Ala Phe Asn Ile Ala Arg 115120 125 Thr Ile Arg Asn Phe Glu Arg Ala Gly Val Ala Ala Val His Ile Glu130 135 140 Asp Gln Val 145 <210> SEQ ID NO 8 <211> LENGTH: 5416 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence; Note = syntheticconstruct <221> NAME/KEY: misc_feature <222> LOCATION: (1)...(5416)<223> OTHER INFORMATION: Note:/ n = a, t, c or g <221> NAME/KEY:misc_feature <222> LOCATION: (1)...(5416) <223> OTHER INFORMATION:Note:/ Seq 4 contains Seq 1 (positions 223- 1407), Seq 2 (positions1921-2880), and Seq 3 (positions 3381-3837) <400> SEQUENCE: 8 tgcaggcatgcaagctggaa ggaaacttgc cgcagccagg aaaacggtgc agtgcaagag 60 agggaagggggcggcggttt gttggcaaga ttgaaacggt ggattgaaaa cagcttctga 120 acaggtggattgccgtttga caggtgagaa gtattttgcc agcagcaaga tacttcttat 180 ataatgaataataatttatt taaaccgtcc tctgaatggg gcgagcagga gtttttgaat 240 ggaatttgtttacgacgtgg cagaatcggc agtcagccct gcggtgatta aagtaatcgg 300 cttgggcggcggcggttgca atgcatccaa taacatggtt gccaacaatg tgcgcggtgt 360 ggagtttatcagtgccaata cggatgcgca gtctctggca aaaaaccatg cggcgaagag 420 aatccagttgggtacgaatc tgacacgcgg tttgggcgcg ggcgcgaatc ccgatatcgg 480 ccgtgcggcagcccaggaag accgggaagc cattgaagaa gccattcgcg gtgcgaatat 540 gctgtttatcacgaccggta tgggcggcgg taccggtacc ggttccgcgc cggttgttgc 600 tgagattgccaagtctttgg gcattctgac cgttgccgtg gttacccgac cgttcgcata 660 tgaaggtaagcgcgtccatg tcgcacaggc agggttggaa cagttgaaag aacacgtcga 720 ttcgctgattatcatcccga acgacaaact gatgactgca ttgggtgaag acgtaacgat 780 gcgcgaagccttccgtgccg ccgacaatgt attgcgcgat gcggtcgcag gcatttccga 840 agtggtaacttgcccgagcg aaatcatcaa cctcgacttt gccgacgtga aaaccgtgat 900 gagcaaccgcggtatcgcta tgatgggttc gggttatgcc caaggtatcg accgtgcgcg 960 tatggcgaccgaccaggcca tttccagtcc gctgctggac gatgtaacct tggacggagc 1020 gcgcggtgtgctggtcaata ttacgactgc tccgggttgc ttgaaaatgt ccgagttgtc 1080 cgaagtcatgaaaatcgtca accaaagcgc gcatcccgat ttggaatgca aattcggtgc 1140 tgctgaagacgagaccatga gcgaagatgc catccggatt accattatcg ctaccggtct 1200 gaaagaaaaaggcgcggtcg attttgttcc ggcaagggag gtagaagcgg ttgccccgtc 1260 caaacaggagcaaagccaca atgtcgaagg tagatccgca ccaatcgcgg tatccgcacg 1320 atgaaccttaccgctgcgga tttcgacaat cagtccgtac ttgacgactt gaaatccctg 1380 cgattttgcgtcgtcaacac aattcagaca aataatgtgc tgtttgcccg taaacctgct 1440 gcctcccgaatcggtttgtc cggtttggga ggtatgtttt tcaagatgtt gcaatttcgt 1500 acggtttgcggtcggcggat tcagattttt ccacttgata cagactttca gatatggaca 1560 cttcaaaacaaacactgttg gacgggattt ttaagctgaa ggcaaacggt acgacggtgc 1620 gtaccgagttgatggcgggt ttgacaactt ttttgacgat gtgctacatc gttaatcgtc 1680 aaccctctgattttgggcga gaccggcatg gatatggggg cggtattcgt cgctacctgt 1740 atcgcgtctgccaatcggct gttttgttat gggttttgtc ggcaactatc cgattgcact 1800 cgcaccggggatggggctga atgcctattt cacctttgcc gtcgttaagg gtatgggctg 1860 ccttggcaggttgcgttggg tgcggtgttc atctccggtc tgattttcat cctgttcagc 1920 ttttttaaagtcagggaaat gctgtcaacg cactgcctat gggtttgaaa atgtcgattg 1980 ctgccggtatcggtttgttt ttggcactga tttccctgaa aggcgcaggc cattatcgtt 2040 gccaatccggcaaccttggt cggtttgggc gatattcatc agccgtccgc gttgttggca 2100 ctgttcggttttgctatggt ggtcgtattg ggacatttcc gcgttcaagg cgcaacatca 2160 tcaccatcttgaccattacc gtcattgcca gcctgatggg tttgaatgaa tttcacggca 2220 tcatcggcgaagtaccgagc attgcgccga cttttatgca gatggatttt gaaggcctgt 2280 ttaccgtcagctggtcagtg attttcgtct tcttcttggt cgatctattt gacagtaccg 2340 gaacgctggtcggcatatcc caccgtgccg ggctgctggt ggacggtaag ctgccccgcc 2400 tgaaacgcgcactgcttgca gactctaccg ccattatggc aggtgcggct ttgggtactt 2460 cttccaccacgccttatgtg gaaagcgcgg cgggcgtatc ggcaggcgga cggaccggcc 2520 tgacggcggttaccgtcggc gtattgatgc tcgcctgcct gatgttttca cctttggcga 2580 aaagtgttcccgcttttggc accgcgcccg ccctgcttta tgtcggcacg cagatgctcc 2640 gcagtgcgagggatattgat tgggacgata tgacggaagc cgcacccgca ttcctgacca 2700 ttgtcttcatgccgtttacc tattcgattg cagacggcat cgccttcggc ttcatcagct 2760 atgccgtggttaaactttta tgccgccgca ccaaagacgt tccgcctatg gaatgggttg 2820 ttgccgtattgtgggcactg aaattctggt atttgggctg attgattcga tattaaaaat 2880 gccgtctgaaaggttttcag acggcatttt gtttgccgat atattaattt ttattaaatt 2940 atataaaaatcaaatacata ataaaataca tcggattgct taaaaataat acattgtttt 3000 ttatgtataaaatattttat aagttttcag gatttggatt attgaaaatt tttcttgatt 3060 tcctgacaattttattgaaa caaataattc aaaattaatc tagtttaatc atagaattaa 3120 aataaaatattaaaattatg taatgagtct ccttaaaaat gtttgacatt ttcagtcttg 3180 tgttttagattatcgaaaaa taaaactaca taacactaca aaggaatatt actatgaaac 3240 caattcagatgttttcccct tttctgaata atccccttgt tttcttcttg tctgcggttt 3300 tgccgcataattccgaacgg tctgctgttt ttctttgatt cgttttaaat atcaataaga 3360 taatttttcccatatatttt taatgattgg attgggatgc ccgacgcgtc ggatggctgt 3420 gttttgccgtccgaatgtga tggaagcctg tccatactga aaaaaagtct ataaaggaga 3480 aatatgatgagtcaacactc tgccggagca cgtttccgcc aagccgtgaa agaatcgaat 3540 ccgcttgccgtcgccggttg cgtcaatgct tattttgcac gattggccac ccaaagcggt 3600 ttcaaagccatctatctgtc tggcggcggc gtggcagcct gttcttgcgg tatccctgat 3660 ttgggcattaccacaatgga agatgtgctg atcgacgcac gacgcattac ggacaacgtg 3720 gatncgcctctgctggtgga catcgatgtg ggttggggcg gtgcattcaa tattgcccgt 3780 accattcgcaactttgaacg cgccggtgtt gcagcggttc acatcgaaga tcaggtagcg 3840 caaaaacgctgcggtcaccg tccgaacaaa gccattgtta tctnaagatg naatggtcga 3900 ccgtatcaaagctgccgtag atgcgcgcgt tgntgngaac ttcgtgatta tggcgcgtac 3960 cgatgcgctggcggtagaag gtttggatgc cgctatcgaa cgcgcccaag cttgtgtcga 4020 aagccggtgcggacatgatt ttccctgaag ccatgaccga tttgaacatg taccgccaat 4080 ttgcagatgcggtgaaagtg cgtgttggcg aacattaccg agtttggttc cactccgctt 4140 tatacccaaagcgagctggc tgaaaacggc gtgtcgctgg tgctgtatcc gctgtcatcg 4200 ttccgtgcagcaagcaaagc cgctctgaat gtttacgaag cgattatgcg cgatggcact 4260 caggcggcggtggtggacag tatgcaaacc cgtgccgagc tgtacgagca tctgaactat 4320 catgccttcgagcaaaaact ggataaattg tttcaaaaat gatttaccgc tttcagacgg 4380 tctttcaacaaatccgcatc ggtcgtctga aaacccgaaa cccataaaaa cacaaaggag 4440 aaataccatgactgaaacta ctcaaacccc gaccttcaaa cctaagaaat ccgttgcgct 4500 ttcaggcgttgcggccggta ataccgcttt gtgtaccgtt ggccgcaccc ggcaacgatt 4560 tggagctatcgcggttacga catcttggat ttgggcacaa aaatgcgttt gaagaagtag 4620 cccacctgctgattcacggt catctgccca acaaattcga cgtggaagct tataaaagga 4680 agctcaaatccatgcgcggc ctgcctatcc gtgtattaaa gttttgggaa agcctgcctg 4740 cacatacccatccggatgga cggtaatggc gtaccggcgg tatccatgct gggctgcgtt 4800 catcccgaacgtgaaagcca tcccggaaag tgaagcgcgc gacatcgccg acaaactgat 4860 tgcagcctcggagcctcctg ctgtactngg tatcaatatc gcacaacggc aaacgcattg 4920 agttgaagcgacgagagaca tcggcggtca tttcctgcaa ctgttncacg gcaacgccca 4980 agcgatcacacatcaaagcc atgcacgttt cactgattct gtatgcgaac acgagttcaa 5040 cgttctacctttaccgtttg ccgttcttct ggtcggttct agccctgtaa aaagagaagg 5100 ttgttagctggcgaaggttt gcagccgtta cagtttcccg cgttatagcg gccaagaaac 5160 gagtttggcgcacggtgaga attacctgtt gcaacgcccc agcctttacc atatgtgggc 5220 ctactggcttnggctagtgc taagaaacgc ggctatgcta gcgcctacat gccgagtgac 5280 gagcgtnacgccatcgcaaa acttatacgc atttcgggaa gccaancgct ggcggcacaa 5340 agcctggatagttgtgcggc taacgnggcc attacgacct catgtatagt cctctgacat 5400 ggcgctanttgcgccc 5416 <210> SEQ ID NO 9 <211> LENGTH: 16 <212> TYPE: RNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence; Note = synthetic construct <221>NAME/KEY: misc_feature <222> LOCATION: (1)...(16) <223> OTHERINFORMATION: Note:/ n = g, a, c or t(u) <221> NAME/KEY: misc_feature<222> LOCATION: (1)...(16) <223> OTHER INFORMATION: Note:/ = consensustarget sequence for hairpin ribozyme <400> SEQUENCE: 9 nnnsngucnn nnnnnn16 <210> SEQ ID NO 10 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence; Note = synthetic construct <221> NAME/KEY:misc_feature <222> LOCATION: (1)...(23) <223> OTHER INFORMATION: Note:/= oligonucleotide HEIC1 <400> SEQUENCE: 10 agcttgaggt cgacgggata tcg 23<210> SEQ ID NO 11 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence; Note = synthetic construct <221> NAME/KEY:misc_feature <222> LOCATION: (1)...(23) <223> OTHER INFORMATION: Note:/= oligonucleotide HEIC2 <400> SEQUENCE: 11 aattcgatat cccgtcgacc tca 23<210> SEQ ID NO 12 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence; Note = synthetic construct <221> NAME/KEY:misc_feature <222> LOCATION: (1)...(20) <223> OTHER INFORMATION: Note:/= primer B800F1 <400> SEQUENCE: 12 cacataaggc gtggtggaag 20 <210> SEQ IDNO 13 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence; Note = synthetic construct <221> NAME/KEY:misc_feature <222> LOCATION: (1)...(20) <223> OTHER INFORMATION: Note:/= target sequence for primer 800F8 <400> SEQUENCE: 13 ctcccaaaccggacaaaccg 20 <210> SEQ ID NO 14 <211> LENGTH: 22 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence; Note = synthetic construct <221>NAME/KEY: misc_feature <222> LOCATION: (1)...(22) <223> OTHERINFORMATION: Note:/ = target sequence for primer 800F9 <400> SEQUENCE:14 gtcaagtacg gactgattgt cg 22 <210> SEQ ID NO 15 <211> LENGTH: 7 <212>TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence; Note = syntheticconstruct <400> SEQUENCE: 15 Gly Gly Gly Thr Gly Thr Gly 1 5

What is claimed is:
 1. An isolated nucleic acid encoding a polypeptideselected from the group of polypeptides consisting of SEQ ID NO:2, SEQID NO:4, SEQ ID NO:5 and SEQ ID NO:7.
 2. The nucleic acid of claim 1,wherein the nucleic acid is selected from the group consisting of SEQ IDNO:1, SEQ ID NO:3 and SEQ ID NO:6.
 3. An isolated nucleic acid whichhybridizes under high stringency conditions to a nucleic acid selectedfrom the group consisting of SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:6wherein the high stringency wash conditions are selected to be about 5°C. lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH.
 4. The isolated nucleicacid of claim 3 wherein the nucleic acid is at least 20 nucleotides inlength.
 5. An isolated nucleic acid which hybridizes under highstringency conditions to SEQ ID NO:8 wherein the stringency washconditions are selected to be about 5° C. lower than the thermal meltingpoint (T_(m)) for the specific sequence at a defined ionic strength andpH.
 6. A recombinant expression vector comprising the nucleic acid ofclaim 5 operably linked to a promoter.
 7. The nucleic acid of claim 6,wherein the nucleic acid, when transduced into a cell, is expressedunder suitable conditions to produce a polypeptide selected from thegroup of polypeptides consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:5, and SEQ ID NO:7.